Glycoprotein VI and uses thereof

ABSTRACT

The invention provides isolated nucleic acid molecules and polypeptide molecules that encode glycoprotein VI, a platelet membrane glycoprotein that is involved platelet-collagen interactions. The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

This application is a divisional of U.S. patent application Ser. No.09/503,387, filed Feb. 14, 2000, pending, which is acontinuation-in-part of U.S. patent application Ser. No. 09/454,824,filed Dec. 6, 1999, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 09/345,068, filed Jun. 30, 1999, nowU.S. Pat. No. 6,245,527, the entire contents of each of theseapplications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The interaction between collagen and platelets is the first event of thenormal hemostatic response to injury. Collagen is the majorextracellular matrix protein present in the subendothelium of bloodvessels. Upon damage to the endothelium lining, as a consequence ofinjury to the vessel wall, collagen fibers, fibrous collagen I and IIIare exposed to platelets. This interaction leads to platelet adhesion,activation with a second phase of adhesion, secretion occurrence, andultimately aggregation and development of a hemostatic plug (Kehrel etal., 1998, Blood 91:491-9).

The mechanism of collagen-platelet interactions is complex. It involves,on one hand, direct binding of collagen to specific platelet receptors(e.g., α₂β₁ integrin, collagen receptor, glycoprotein IV, andglycoprotein VI) and, on the other hand, indirect binding of collagenvia bridging proteins (e.g., von Willebrand Factor (vWF)) that bind toboth collagen and membrane receptors on platelets. Recent reportssupport a two-step mechanism of collagen-platelet interaction,consisting a platelet adhesion followed by platelet activation (Verkleijet al., 1998, Blood 91:3808-16). The first step involves the binding ofcollagen-bound vWF by the platelet receptor complex glycoproteinIb/IX/V, followed by the direct binding of integrin α₂β₁ to collagen(Moroi et al., 1997, Thrombosis and Haemostasis 78:439-444 and Barnes etal., 1998, Current Opinion in Hematology 6:314-320). This step resultsin platelets adhering to the subendothelium of blood vessels underphysiological conditions. The second step of collagen-plateletinteraction involves another platelet collagen receptor, glycoprotein VI(Barnes et al., 1998, Current Opinion in Hematology 6:314-320). Thisbinding leads to strengthening of attachment and platelet activation. Itis believed that glycoprotein VI (GPVI) has a minor importance in thefirst step of adhesion but plays a major role in the second step ofcollagen-platelet interaction resulting in full platelet activation andconsequently the formation of the platelet aggregates (Aria et al.,1995, British J. of Haematology 89:124-130).

Glycoprotein VI

Glycoprotein VI (GPVI) is a platelet membrane glycoprotein that isinvolved in platelet-collagen interactions. In particular, GPVI is atransmembrane collagen receptor expressed on the surface of platelets.GPVI has an apparent molecular mass of 58 kDa in its non-reduced formand 62 kDa after disulfide bond reduction as determined by its migrationvia SDS-PAGE. Treatment of platelets with N-glycanase has been shown toresult in a faster migration of GPVI in SDS-PAGE by two kDa, whichprobably corresponds to only one N-glycosylation site.

The existence of a 62 kDa protein, later identified as GPVI, was firstdetected as an antigen recognized by the sera of a patient withsteroid-responsive immune thrombocytopenic purpura associated withdefective collagen-induced platelet functions (Sugiyama et al., 1987,Blood 69: 1712-1720). The patient's plasma, as well as a preparation offull length IgG antibodies, induced irreversible aggregation and ATPrelease in normal platelet-rich plasma. However, Fab fragments preparedfrom the serum of this patient blocked platelet aggregation induced bycollagen (Sugiyama et al., 1987, Blood 69: 1712-172).

The importance of GPVI in platelet/collagen interactions was furtherconfirmed by comparing the expression of platelet collagen receptorsfrom a different patient, with a mild bleeding disorder, to that of anormal individual (Moroi et al., 1989, J. Clin. Invest. 84(5):1440-5).The patient's platelets lacked collagen-induced aggregation andadhesion, but retained normal aggregation and release by other agonists.The expression of a 61 kDa membrane glycoprotein was detected onnon-reduced, two-dimensional SDS-PAGE, but was reduced compared to theexpression levels found in a normal individual. This glycoprotein wastermed glycoprotein VI (GPVI). The patient's platelets did not bind totypes I and III collagen fibrils suggesting that GPVI functions as acollagen receptor involved in collagen-induced platelet activation andaggregation.

GPVI has been shown to be constitutively associated with the Fc receptorgamma (FcRγ), and FcRγ expression is lacking in GPVI-deficientplatelets, suggesting that GPVI and FcRγ are co-expressed in platelets(Tsuji et al., 1997, J. Biol. Chem. 272:23528-31). Further,cross-linking of GPVI by F(ab′)2 fragments of anti-GPVI IgG has beenshown to result in the tyrosine phosphorylation of the FcRγ-chain. FcRγis tyrosine-phosphorylated upon platelet activation by collagen,collagen related peptide (CRP; Gibbins et al., 1997, FEBS Lett.413:255-259) or the snake venom component convulxin that acts as aplatelet agonist (Cvx; Lagrue et al., 1999, FEBS Letts. 448:95-100).Phosphorylation occurs on the immunoreceptor tyrosine-based activationmotifs (ITAM) of FcRγ by kinases of the Src family (p59Fyn and p53/56lyn) (Briddon S J and Watson, 1999, Biochem J. 338:203-9).Phosphorylation of FcRγ allows Syk, a signaling molecule, to bind and tobe in turn phosphorylated and to activate phospholipase Cγ2 (PLCγ2).Further, platelet stimulation by collagen or Cvx have been shown toinvolve the association of phosphatidylinositol 3-kinase (PI3 kinase)and the adapter protein linker for activator of T cells (LAT) to theFcRγ (Carlsson et al., 1998, Blood 92:1526-31). Thus, FcRγ appears tointeract with GPVI to effect signaling.

The results from the GPVI signal transduction pathway activation studiesperformed suggest that strong similarities exist between the GPVIsignaling pathway in platelets and the one used by receptors for immunecomplexes, such as the high-affinity and low affinity receptors for IgG(FcRγI and FcRγIII), the high-affinity receptor for IgE (FcRεI) and thereceptor for IgA (FcRαI) (Maliszewski et al., 1990, J. Exp. Med.172:1665-72). These receptors also signal via the FcRγ chain and Syk.Expression of the FcRγI, FcRγIII has not been reported in platelets. TheFcRγIIa seems to be the only IgG Fc-receptor consistently expressed onplatelets, and it contains one ITAM. This receptor has been suggested tobe involved in thrombocytopenia and thromboembolic complications ofheparin-induced thrombocytopenia (HIT), the most common drug-inducedimmune thrombocytopenia (Carlsson et al., 1998, Blood 92:1526-31) andmay also be involved in other immune thrombocytopenia such as immunethrombocytopenia purpura (Loscalzo, J., and Schafer, A. I., 1998,Thrombosis and Hemorrhage, J. Loscalzo and A. I. Schafer, eds.,Baltimore: Williams and Wilkins).

Since its detection, the function of GPVI in platelet-collageninteractions and the signal transduction pathway induced by GPVI hasbeen studied. However, the molecular cloning of GPVI has been elusivedue, at least in part, to its extensive O-linked glycosylation. Theinability to clone GPVI has limited the experiments that can beperformed to better understand the role of GPVI in collagen-inducedplatelet activation and aggregation. Further, the development oftreatments for disorders, such as bleeding disorders, resulting frommutations in GPVI or its promoter, have been hindered by the lack ofknowledge about the nucleic acid and amino acid sequences of GPVI.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofcDNA molecules which encode the TANGO 268 proteins, all of which aretransmembrane proteins.

In particular, TANGO 268 represents the platelet-expressed collagenreceptor GPVI. This conclusion is based, at least in part, on thefollowing evidence: (1) the glycosylated molecular weights of TANGO 268and GPVI are identical or similar; (2) TANGO 268 and GPVI are bothrecognized by anti-GPVI antibodies and bind to Cvx; (3) TANGO 268 andGPVI are both preferentially expressed in the megakaryocytic cells; (4)TANGO 268 and GPVI are both predicted to have a single N-glycosylationsite; (5) the molecular mass of the 40 kDa unglycosylated TANGO 268 ispredicted to be approximately 62 kDa, the apparent molecular mass ofGPVI, upon N— and O-linked glycosylation; (6) the presence of twoimmunoglobulin-like domains in TANGO 268 indicates that, like GPVI TANGO268 interacts with the FcRγ; (7) the absence of a large intracytoplasmictail, suggesting that this membrane-bound glycoprotein has no signalingrole but associates with another member of the Ig family (e.g., FcRγ)protein to transduce a signal; and (8) the presence of a charged residue(arginine) in the transmembrane domain of TANGO 268 which is predictedto be present in GPVI based on its association with the FcRγ.

The TANGO 268 proteins are members of the Ig superfamily. The TANGO 268proteins, fragments, derivatives, and variants thereof are collectivelyreferred to herein as “polypeptides of the invention” or “proteins ofthe invention.” Nucleic acid molecules encoding the polypeptides orproteins of the invention are collectively referred to as “nucleic acidsof the invention.”

The nucleic acids and polypeptides of the present invention are usefulas modulating agents in regulating a variety of cellular processes.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding a polypeptide of the invention or a biologicallyactive portion thereof. The present invention also provides nucleic acidmolecules which are suitable for use as primers or hybridization probesfor the detection of nucleic acids encoding a polypeptide of theinvention.

The invention features nucleic acid molecules which are at least 50%,55%, 65%, 75%,85%, 95%, or 98% identical to the nucleotide sequence ofSEQ ID NO:1 the nucleotide sequence of the cDNA insert of an EpthEa11d1clone deposited with ATCC as Accession Number 207180, or a complementthereof.

The invention features nucleic acid molecules which are at least 40%,45%, 50%, 55%,65%, 75%, 85%, 95%, or 98% identical to the nucleotidesequence of SEQ ID NO:2 the nucleotide sequence of the cDNA insert of anEpthEa11d1 clone deposited with ATCC as Accession Number 207180, or acomplement thereof.

The invention features nucleic acid molecules which are at least 40%,45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the nucleotidesequence of SEQ ID NO:14 the nucleotide sequence of the cDNA insert ofan EpTm268 clone deposited with ATCC as PTA-225, or a complementthereof.

The invention features nucleic acid molecules which are at least 35%,40%, 45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to thenucleotide sequence of SEQ ID NO:15 the nucleotide sequence of the cDNAinsert of an EpTm268 clone deposited with ATCC as PTA-225, or acomplement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900 or 2000 nucleotides of the nucleotide sequence ofSEQ ID NO:1 the nucleotide sequence of an EpthEa11d1 cDNA of ATCCAccession Number 207180, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 950 or 1000nucleotides of the nucleotide sequence of SEQ ID NO:2, or a complementthereof.

The invention features nucleic acid molecules which include a fragmentof at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or1100 nucleotides of the nucleotide sequence of SEQ ID NO:14 thenucleotide sequence of an EpTm268 cDNA of ATCC PTA-225, or a complementthereof.

The invention features nucleic acid molecules which include a fragmentof at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 950 or 1000nucleotides of the nucleotide sequence of SEQ ID NO:15, or a complementthereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%,or 98% identical to the amino acid sequence of SEQ ID NO:3, the aminoacid sequence encoded by an EpthEa11d1 cDNA of ATCC Accession Number207180, or a complement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%,or 98% identical to the amino acid sequence of SEQ ID NO:16, the aminoacid sequence encoded by an EpTm268 cDNA of ATCC PTA-225, or acomplement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%,or 98% identical to the amino acid sequence of SEQ ID NO:3 or 16, theamino acid sequence encoded by EpthEa11d1 or EpTm268 of ATCC AccessionNumber 207180 or PTA-225, or a complement thereof, wherein the proteinencoded by the nucleotide sequence also exhibits at least one structuraland/or functional feature of a polypeptide of the invention.

In preferred embodiments, the nucleic acid molecules have the nucleotidesequence of SEQ ID NO:1, 2, 14, 15 or the nucleotide sequence of thecDNA clones of ATCC Accession Number 207180 or PTA-225.

The invention also includes nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ ID NO:3,or a fragment including at least 15, 25, 30, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 315 or 330 contiguous amino acids of SEQ IDNO:3, or the amino acid sequence encoded by an EpthEa11d1 cDNA of ATCCAccession Number 207180.

The invention also includes nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:16, or a fragment including at least 15, 25, 30, 50, 75, 100, 125,150, 175, 200, 225, 250, 275 or 300 contiguous amino acids of SEQ IDNO:16, or the amino acid sequence encoded by an EpTm268 cDNA of ATCCPTA-225.

The invention also includes nucleic acid molecules which encode anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:3 or 16, or the amino acid sequenceencoded by a cDNA of ATCC Accession Number 207180 or PTA-225, whereinthe nucleic acid molecule hybridizes to a nucleic acid moleculeconsisting of a nucleic acid sequence encoding SEQ ID NO:3 or 16, or theamino acid sequence encoded by a cDNA of ATCC Accession Number 207180 orPTA-225, or a complement thereof under stringent conditions.

The invention also includes isolated polypeptides or proteins having anamino acid sequence that is at least about 30%, preferably 35%, 45%,55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence ofSEQ ID NO:3, or the amino acid sequence encoded by an EpthEal ldl cDNAof ATCC Accession Number 207180.

The invention also includes isolated polypeptides or proteins having anamino acid sequence that is at least about 30%, preferably 35%, 40%,45%, 50%, 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:16, or the amino acid sequence encoded by anEpTm268 cDNA of ATCC PTA-225.

The invention also features nucleic acid molecules which encode apolypeptide fragment of at least 15, 25, 30, 50, 75, 100, 125, 150, 175,200 or more contiguous amino acids of SEQ ID NO:3 or 16, or the aminoacid sequence encoded by EpthEa11d1 or EpTm268 of ATCC Accession Number207180 or PTA-225, respectively, wherein the fragment also exhibits atleast one structural and/or functional feature of a polypeptide of theinvention.

The invention also features isolated polypeptides or proteins having anamino acid sequence that is at least about 30%, preferably 35%, 40%,45%, 50%, 55%, 65%, 75%, 85%, 95% or 98% identical to the amino acidsequence of SEQ ID NO:3 or 16, or the amino acid sequence encoded byEpthEa11a1 or EpTm268 of Accession Number 207180 or PTA-225,respectively, wherein the protein or polypeptides also exhibit at leastone structural and/or functional feature of a polypeptide of theinvention.

The invention also includes isolated polypeptides or proteins which areencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 50%, preferably 55%, 60%, 65%, 75%, 85%, 95% or 98%identical to the nucleic acid sequence encoding SEQ ID NO:3, andisolated polypeptides or proteins which are encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 or 2, a complement thereof, or thenon-coding strand of an EpthEa11d1 cDNA of ATCC Accession Number 207180.

The invention also includes isolated polypeptides or proteins which areencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 35%, preferably 40%, 45%, 50%, 55%, 60%, 65%, 75%, 85%,95% or 98% identical to the nucleic acid sequence encoding SEQ ID NO:16,and isolated polypeptides or proteins which are encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:14 or 15, a complement thereof, or thenon-coding strand of an EpTm268 cDNA of ATCC PTA-225.

The invention also features isolated polypeptides or proteins which areencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 30%, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%,85%, 95% or 98% identical to a nucleic acid sequence encoding SEQ IDNO:3 or 16, isolated polypeptides or proteins which are encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, 2, 14 or 15, a complementthereof, or the non-coding strand of EpthEa11d1 or EpTm268 of ATCCAccession Number 207180 or PTA-225, respectively, wherein thepolypeptides or proteins also exhibit at least one structural and/orfunctional feature of a polypeptide of the invention.

The invention also includes polypeptides which are naturally occurringallelic variants of a polypeptide that includes the amino acid sequenceof SEQ ID NO:3 or 16, or the amino acid sequence encoded by a cDNA ofATCC Accession Number 207180, wherein the polypeptide is encoded by anucleic acid molecule which hybridizes to a nucleic acid molecule havingthe sequence of SEQ ID NO:1, 2, 14, 15, or a complement thereof understringent conditions.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1 or 2, an EpthEa11d1 cDNA of ATCC AccessionNumber 207180, or a complement thereof. In other embodiments, thenucleic acid molecules are at least 480, 500, 530, 550, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000nucleotides in length and hybridize under stringent conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,an EpthEa11d1 cDNA of ATCC Accession Number 207180, or a complementthereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:14 or 15, an EpTm268 cDNA of ATCC PTA-225, or acomplement thereof. In other embodiments, the nucleic acid molecules areat least 400, 450, 500, 530, 550, 600, 700, 800, 900, 1000, 1100 or 1150nucleotides in length and hybridize under stringent conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:14, an EpTm268 cDNA of ATCC PTA-225, or a complement thereof.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1, 2, 14 or 15, or a nucleotide sequence ofEpthEa11d1 or EpTm268 of ATCC Accession Number 207180 or PTA-225, orcomplement thereof, wherein such nucleic acid molecules encodepolypeptides or proteins that exhibit at least one structural and/orfunctional feature of a polypeptide of the invention.

In one embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a nucleic acid ofthe invention.

Another aspect of the invention provides vectors, e.g., recombinantexpression vectors, comprising a nucleic acid molecule of the invention.In another embodiment, the invention provides host cells containing sucha vector or engineered to contain and/or express a nucleic acid moleculeof the invention. The invention also provides methods for producing apolypeptide of the invention by culturing, in a suitable medium, a hostcell of the invention such that a polypeptide of the invention isproduced.

Another aspect of this invention features isolated or recombinantproteins and polypeptides of the invention. Preferred proteins andpolypeptides possess at least one biological activity possessed by thecorresponding naturally-occurring human polypeptide. An activity, abiological activity, or a functional activity of a polypeptide ornucleic acid of the invention refers to an activity exerted by aprotein, polypeptide or nucleic acid molecule of the invention on aresponsive cell as determined in vivo or in vitro, according to standardtechniques. Such activities can be a direct activity, such as anassociation with or an enzymatic activity on a second protein, or anindirect activity, such as a cellular signaling activity mediated byinteraction of the protein with a second protein.

For TANGO 268, biological activities include, e.g., (1) the ability tomodulate, e.g., stabilize, promote, inhibit or disrupt protein-proteininteractions (e.g., homophilic and/or heterophilic), and protein-ligandinteractions, e.g., in receptor-ligand recognition; (2) the ability tomodulate cell-cell interactions and/or cell-extracellular matrix (ECM)interactions, e.g., by modulating platelet interactions withsubendothelial components, e.g., collagen, integrins and other ECMproteins; (3) the ability to modulate the host immune response, e.g., bymodulating one or more elements in the inflammatory response; (4) theability to modulate the proliferation, differentiation and/or activityof megakaryocytes and/or platelets; (5) the ability to modulateintracellular signaling cascades (e.g., signal transduction cascades);(6) the ability to modulate immunoregulatory functions; (7) the abilityto modulate platelet morphology, migration, aggregation, degranulationand/or function; (8) the ability to interact with (e.g., bind todirectly or indirectly, for example, as part of a complex comprisingTANGO 268) one or more collagen molecules; (9) the ability to modulatecollagen binding to platelets; (9) the ability to mediate and/ormodulate intracellular Ca²⁺ levels, metabolism and/or turnover ofphosphatidylinositides, and phosphorylation of proteins (e.g., c-Src,Syk, PLCγ2 and/or FcRγ) via, for example, their tyrosine residues; (10)the ability mediate and/or modulate collagen-induced platelet adhesionand aggregation (e.g., thrombus formation), for example, in mediatingand/or modulating secretion of the contents of platelet granules; (11)the ability mediate and/or modulate platelet adhesion and aggregation;(12) the ability to interact with (e.g., bind to directly or indirectly,for example, as part of a complex comprising TANGO 268) convulxin; (13)the ability to bind to an antibody from a patient with idiopathicthrombocytopenic purpura (ITP); (14) the ability to associate and/orco-express with FcRγ, e.g., FcRγ in platelets; (15) the ability toinduce and/or modulate tumor formation, tumor cell migration, and/ortumor cell metastasis; and (16) the ability to induce and/or modulatecoronary diseases (e.g., atherosclerosis).

In one embodiment, a polypeptide of the invention has an amino acidsequence sufficiently identical to an identified domain of a polypeptideof the invention. As used herein, the term “sufficiently identical”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., with asimilar side chain) amino acid residues or nucleotides to a second aminoacid or nucleotide sequence such that the first and second amino acid ornucleotide sequences have or encode a common structural domain and/orcommon functional activity. For example, amino acid or nucleotidesequences which contain or encode a common structural domain havingabout 60% identity, preferably 65% identity, more preferably 75%, 85%,95%, 98% or more identity are defined herein as sufficiently identical.

In one embodiment a 268 protein includes at least one or more of thefollowing domains: a signal sequence, an extracellular domain, animmunoglobulin-like domain, a transmembrane domain, and an intracellulardomain.

The polypeptides of the present invention, or biologically activeportions thereof, can be operably linked to a heterologous amino acidsequence to form fusion proteins. The invention further featuresantibodies, such as monoclonal or polyclonal antibodies, thatspecifically bind a polypeptide of the invention. In addition, thepolypeptides of the invention or biologically active portions thereofcan be incorporated into pharmaceutical compositions, which optionallyinclude pharmaceutically acceptable carriers.

In another aspect, the present invention provides methods for detectingthe presence, activity or expression of a polypeptide of the inventionin a biological sample by contacting the biological sample with an agentcapable of detecting an indicator of the presence, activity orexpression such that the presence activity or expression of apolypeptide of the invention in the biological sample.

In another aspect, the invention provides methods for modulatingactivity of a polypeptide of the invention comprising contacting a cellwith an agent that modulates (inhibits or stimulates) the activity orexpression of a polypeptide of the invention such that activity orexpression in the cell is modulated. In one embodiment, the agent is anantibody that specifically binds to a polypeptide of the invention.

In another embodiment, the agent modulates expression of a polypeptideof the invention by modulating transcription, splicing, or translationof an mRNA encoding a polypeptide of the invention. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of an mRNA encoding apolypeptide of the invention.

In yet a further aspect, the invention provides substantially purifiedantibodies or fragment thereof, and non-human antibodies or fragmentsthereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:3 or 16, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 207180, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as PTA-225; afragment of at least 15 amino acid residues of the amino acid sequenceof SEQ ID NO:3 or 16; an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO:3 or 16, wherein thepercent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4; and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to the nucleicacid molecule consisting of SEQ ID NO:1, 2, 14, or 15 under conditionsof hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at65° C. In various embodiments, the substantially purified antibodies ofthe invention, or fragments thereof, can be human, non-human, chimericand/or humanized antibodies.

In another aspect, the invention provides non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:3 or 16, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 207180, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as PTA-225; afragment of at least 15 amino acid residues of the amino acid sequenceof SEQ ID NO:3 or 16; an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO:3 or 16, wherein thepercent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4; and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to the nucleicacid molecule consisting of SEQ ID NO:1, 2, 14, or 15 under conditionsof hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at65° C. Such non-human antibodies can be goat, mouse, sheep, horse,chicken, rabbit, or rat antibodies. Alternatively, the non-humanantibodies of the invention can be chimeric and/or humanized antibodies.In addition, the non-human antibodies of the invention can be polyclonalantibodies or monoclonal antibodies.

In still a further aspect, the invention provides monoclonal antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:3 or 16, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 207180, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as PTA-225; afragment of at least 15 amino acid residues of the amino acid sequenceof SEQ ID NO:3 or 16; an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO:3 or 16, wherein thepercent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4; and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to the nucleicacid molecule consisting of SEQ ID NO:1, 2, 14, or 15 under conditionsof hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at65° C. The monoclonal antibodies can be human, humanized, chimericand/or non-human antibodies.

In a particularly preferred embodiment, the substantially purifiedantibodies or fragments thereof, the non-human antibodies or fragmentsthereof, and/or the monoclonal antibodies or fragments thereof, of theinvention specifically bind to an extracellular domain of the amino acidsequence of SEQ ID NO:3 or 16. Preferably, the extracellular domain towhich the antibody, or fragment thereof, binds comprises amino acidresidues 21 to 269 of SEQ ID NO:3 or amino acid residues 22 to 267 ofSEQ ID NO:16. In an alternative embodiment, the extracellular domain towhich the substantially purified antibody binds comprises animmunoglobulin-like domain. In one aspect, such an immunoglobulin-likedomain comprises amino acid residues 48 to 88 or 134 to 180 of SEQ IDNO:3 or amino acid residues 49 to 89 or 135 to 181 of SEQ ID NO:16.

Any of the antibodies of the invention can be conjugated to atherapeutic moiety or to a detectable substance. Non-limiting examplesof detectable substances that can be conjugated to the antibodies of theinvention are an enzyme, a prosthetic group, a fluorescent material, aluminescent material, a bioluminescent material, and a radioactivematerial.

The invention also provides a kit containing an antibody of theinvention conjugated to a detectable substance, and instructions foruse. Still another aspect of the invention is a pharmaceuticalcomposition comprising an antibody of the invention and apharmaceutically acceptable carrier. In preferred embodiments, thepharmaceutical composition contains an antibody of the invention, atherapeutic moiety, and a pharmaceutically acceptable carrier.

Still another aspect of the invention is a method of making an antibodythat specifically recognizes GPVI, the method comprising immunizing amammal with a polypeptide. The polypeptide used as an immungen comprisesan amino acid sequence selected from the group consisting of: the aminoacid sequence of SEQ ID NO:3 or 16, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as AccessionNumber 207180, or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with ATCC as PTA-225; a fragment of at least 15amino acid residues of the amino acid sequence of SEQ ID NO:3 or 16; anamino acid sequence which is at least 95% identical to the amino acidsequence of SEQ ID NO:3 or 16, wherein the percent identity isdetermined using the ALIGN program of the GCG software package with aPAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4; and an amino acid sequence which is encoded by a nucleicacid molecule which hybridizes to the nucleic acid molecule consistingof SEQ ID NO:1, 2, 14, or 15 under conditions of hybridization of 6×SSCat 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. After immunization,a sample is collected from the mammal that contains an antibody thatspecifically recognizes GPVI. Preferably, the polypeptide isrecombinantly produced using a non-human host cell. Optionally, theantibodies can be further purified from the sample using techniques wellknown to those of skill in the art. The method can further compriseproducing a monoclonal antibody-producing cell from the cells of themammal. Optionally, antibodies are collected from the antibody-producingcell.

The present invention also provides methods to treat a subject having adisorder characterized by aberrant activity of a polypeptide of theinvention or aberrant expression of a nucleic acid of the invention byadministering an agent which is a modulator of the activity of apolypeptide of the invention or a modulator of the expression of anucleic acid of the invention to the subject. In one embodiment, themodulator is a protein of the invention. In another embodiment, themodulator is a nucleic acid of the invention. In other embodiments, themodulator is a peptide, peptidomimetic, or other small molecule.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a polypeptide of the invention, (ii) mis-regulation of a geneencoding a polypeptide of the invention, and (iii) aberrantpost-translational modification of the invention wherein a wild-typeform of the gene encodes a protein having the activity of thepolypeptide of the invention.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a polypeptide of theinvention. In general, such methods entail measuring a biologicalactivity of the polypeptide in the presence and absence of a testcompound and identifying those compounds which alter the activity of thepolypeptide.

The invention also features methods for identifying a compound whichmodulates the expression of a polypeptide or nucleic acid of theinvention by measuring the expression of the polypeptide or nucleic acidin the presence and absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict the cDNA sequence of human TANGO 268 (SEQ ID NO:1) andthe predicted amino acid sequence of human TANGO 268 (SEQ ID NO:3). Theopen reading frame of SEQ ID NO:1 extends from nucleotide 36 tonucleotide 1052 of SEQ ID NO:1 (SEQ ID NO:2).

FIG. 2 depicts a hydropathy plot of human TANGO 268. Relativelyhydrophobic regions of the protein are above the dashed horizontal line,and relatively hydrophilic regions of the protein are below the dashedhorizontal line. The cysteine residues (cys) are indicated by shortvertical lines just below the hydropathy trace. The dashed vertical lineseparates the signal sequence (amino acids 1 to 20 of SEQ ID NO:3; SEQID NO:4) on the left from the mature protein (amino acids 21 to 339 ofSEQ ID NO:3; SEQ ID NO:5) on the right. Below the hydropathy plot, theamino acid sequence of human TANGO 268 is depicted.

FIGS. 3A-C depict an alignment of the nucleotide sequence of the openreading frame for human monocyte inhibitory receptor precursor (SEQ IDNO:24; GenBank Accession Number U91928) and the nucleotide sequence ofthe open reading frame for human TANGO 268 (SEQ ID NO:2). The nucleotidesequences of coding regions of human monocyte inhibitory receptorprecursor and human TANGO 268 are 37.7% identical. The nucleotidesequences of full-length, including the 5′ and 3′ untranslated regions(UTRs), human monocyte inhibitory receptor precursor SEQ ID NO:11;GenBank Accession Number U91928) and human TANGO 268 are 49.9%identical. These alignments were performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIG. 4 depicts an alignment of the amino acid sequence of human monocyteinhibitory receptor precursor (SEQ ID NO:12) and the amino acid sequenceof human TANGO 268 (SEQ ID NO:3). The amino acid sequences of humanmonocyte inhibitory receptor precursor and human TANGO 268 are 23.0%identical. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIG. 5A depicts an alignment of the amino acid sequence of a typicalimmunoglobulin domain (SEQ ID NO:13; GenBank Accession Number PF00047)and amino acid residues 41 to 90 of human TANGO 268 (SEQ ID NO:3). Thisalignments was performed using the ALIGN alignment program with a PAM120scoring matrix, a gap length penalty of 12, and a gap penalty of 4.

FIG. 5B depicts an alignment of the amino acid sequence of a typicalimmunoglobulin domain (SEQ ID NO:13; GenBank Accession Number PF00047)and amino acid residues 127 to 182 of human TANGO 268 (SEQ ID NO:3).This alignment was performed using the ALIGN alignment program with aPAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of4.

FIG. 6 depicts a cDNA sequence of mouse TANGO 268 (SEQ ID NO:14) and thepredicted amino acid sequence of mouse TANGO 268 (SEQ ID NO:15). Theopen reading frame of SEQ ID NO:14 extends from nucleotide 63 to 1001 ofSEQ ID NO:14 (SEQ ID NO:15).

FIG. 7 depicts a hydropathy plot of mouse TANGO 268. Relativelyhydrophobic regions of the protein are above the dashed horizontal line,and relatively hydrophilic regions of the protein are below the dashedhorizontal line. The cysteine residues (cys) are indicated by shortvertical lines just below the hydropathy trace. The dashed vertical lineseparates the signal sequence (amino acids 1 to 21 of SEQ ID NO:16; SEQID NO:17) on the left from the mature protein (amino acids 22 to 313 ofSEQ ID NO:16; SEQ ID NO:18) on the right. Below the hydropathy plot, theamino acid sequence of mouse TANGO 268 is depicted.

FIGS. 8A-D depict an alignment of the nucleotide sequence of the openreading frame for human monocyte inhibitory receptor precursor (SEQ IDNO:24; GenBank Accession Number U91928) and the nucleotide sequence ofthe open reading frame for mouse TANGO 268 (SEQ ID NO:15). Thenucleotide sequences of coding regions of human monocyte inhibitoryreceptor precursor and mouse TANGO 268 are 34.4% identical. Thenucleotide sequences of full-length, including the 5′ and 3′untranslated regions (UTRs), human monocyte inhibitory receptorprecursor SEQ ID NO:11; GenBank Accession Number U91928) and mouse TANGO268 are 35.6% identical. These alignments were performed using the ALIGNalignment program with a PAM 120 scoring matrix, a gap length penalty of12, and a gap penalty of 4.

FIG. 9 depicts an alignment of the amino acid sequence of human monocyteinhibitory receptor precursor (SEQ ID NO:12) and the amino acid sequenceof mouse TANGO 268 (SEQ ID NO:16). The amino acid sequences of humanmonocyte inhibitory receptor precursor and mouse TANGO 268 are 20.3%identical. This alignment was performed using the ALIGN alignmentprogram with a PAM120 scoring matrix, a gap length penalty of 12, and agap penalty of 4.

FIG. 10A depicts an alignment of the amino acid sequence ofimmunoglobulin domain (SEQ ID NO:12; GenBank Accession Number PF00047)and amino acid residues 42 to 91 of mouse TANGO 268 (SEQ ID NO:16). Thisalignment was performed using the ALIGN alignment program with a PAM120scoring matrix, a gap length penalty of 12, and a gap penalty of 4.

FIG. 10B depicts an alignment of the amino acid sequence of a typicalimmunoglobulin domain (SEQ ID NO:12; GenBank Accession Number PF00047)and amino acid residues 128 to 183 of mouse TANGO 268 (SEQ ID NO:16).This alignment was performed using the ALIGN alignment program with aPAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of4.

FIG. 11 depicts an alignment of the amino acid sequence of human TANGO268 (SEQ ID NO:3) and the amino acid sequence of mouse TANGO 268 (SEQ IDNO:16). The alignment demonstrates that the amino acid sequences ofhuman and mouse TANGO 268 are 64.4% identical. The alignment wasperformed using the ALIGN program with a PAM120 scoring matrix, a gaplength penalty of 12 and a gap penalty of 4. The sequences within theboxes are the signal sequences for human and mouse TANGO 268; the lineabove the two sequences indicates the Ig-like domains for human andmouse TANGO 268; and the arrow above the sequences points to the chargedresidue (arginine) in human and mouse TANGO 268.

FIG. 12 depicts the results from the ligand blotting assay with¹²⁵I-convulxin (Cvx), demonstrating that TANGO 268 specifically bindsCvx. Lane 1 contains platelet lysate, lane 2 contains lysate fromexpression vector-only transfected CHO cells, and lane 3 contains TANGO268-transfected CHO cell lysate. The cell lysates were separated onpolyacrylamide gels, transferred to PVDF membranes, and the membraneswere incubated with ¹²⁵I-Cvx. The interaction between ¹²⁵I-Cvx and TANGO268 was detected by autoradiography.

FIG. 13A depicts the results from the immunoblotting assay withanti-GPVI Ig antibody, demonstrating that TANGO 268 specifically bindsto anti-GPVI Ig antibody. The cell lysates were separated onpolyacrylamide gels, transferred to PVDF membranes, the membranes wereincubated with anti-GPVI IgG antibody followed by an incubation withperoxidase-coupled protein A, and TANGO 268 expression was detected byenhanced chemiluminescence.

FIG. 13B depicts the results of anti-GPVI IgG binding followingcompetition with Cvx, which demonstrates that Cvx competes withanti-GPVI Ig antibody for binding to TANGO 268. Lane 1 contains plateletlysate, lane 2 contains lysate from expression vector-only transfectedCHO cells, and lane 3 contains TANGO 268-transfected CHO cell lysate.The cell lysates were separated on polyacrylamide gels, transferred toPVDF membranes, the membranes were incubated with anti-GPVI IgG antibodyin the presence of Cvx followed by an incubation with peroxidase-coupledprotein A, and TANGO 268 expression was detected by enhancedchemiluminescence.

FIG. 14: Tissue expression of hGPVI and mGPVI using RT-PCR, northernblot and ISH analysis:

FIG. 14A: In situ hybridization of a day 12.5 mouse embryo.Hybridization is exclusively observed in the liver during embryogenesis.No signal was seen with the sense probe (data not shown). High magnituderesolution shows that the only positive cell population corresponded tofetal megakaryocytes (data not shown). In adult, expression in liver wasno longer observed but a strong, multifocal signal was seen in spleenand in the bone marrow.

FIG. 14B: high magnitude resolution from a photoemulsion processingcarried out on a 6-week-old mouse femur section shows expressionrestricted to megakaryocytes. No signal was observed in any other adulttissues analyzed (see results).

FIG. 14C: RT-PCR analysis from human samples. β₂ microglobulin and GPVItranscripts were co-amplified. The high molecular weight fragment (830bp) is generated from the GPVI primers and the low molecular weightfragment (603 bp) is generated from the β₂ microglobulin primers. The β₂microglobulin PCR product, used as a loading control, is present in allthe samples in similar quantity. In contrast, GPVI is only amplified inmegakaryocyte-enriched samples (adult and newborn), in cell linesdisplaying strong MKC features (HEL, MEG01, DAMI, MO7E, mpl-UT7) and ata lesser extend in fetal liver cells. A very low signal is also detectedin the K562 and KG1 cell lines, two cell lines which also express GPIIbat low level, but no expression was detected in the other samples.

FIG. 14D: Northern blot analysis of human tissues. A 2 kb transcript isonly observed in bone marrow and fetal liver. A signal is also observedwith peripheral blood leucocytes (PBL). However, when the same blot washybridized with a GPIlb probe, a platelet protein absent in PBL,transcripts were also detected suggesting that the signal was due toplatelet RNA contamination. No signal was observed in a different PBLsample but also in brain, heart, skeletal muscle, colon, thymus, spleen,kidney, liver, small intestine, placenta, lung or lymph nodes (data notshown).

FIG. 15: Binding of Cvx to murine hematopoietic cell lines

Hematopoietic cell lines were transduced with a retrovirus expressingrmGPVI. Control cells were transduced with the empty vector. Cells wereincubated with FITC-coupled Cvx or FITC-coupled bothrojaracin as acontrol and analyzed by flow cytometry. FIG. 15A: FDC-P1, FIG. 15B:Ba/F3, FIG. 15C: 32D. Dotted line: control cells transduced with theempty vector, plain line cells transduced with the retrovirus carryingrmGPVI.

FIG. 16: Adhesion of cells expressing rhGPVI or rmGPVI to immobilizedCvx or collagen. BSA, Cvx or collagen type I were immobilized onmicrotitration plates. GPVI transduced or control U937 (FIG. 16A) orFDC-P1 (FIG. 16B) cells were labeled with ⁵¹Cr and incubated for 60 minin the wells. After aspiration of the non-bound cells and washing,radioactivity associated to the wells was counted to determine adherentcell number. Results are expressed as the percentage of the cells addedto the wells and are the mean±SEM of three determinations. Empty bars:control cells; filled bars: GPVI expressing cells.

FIG. 17: Coexpression of recombinant human GPVI with FcRγ chain.

Lysates from GPVI transduced or control U937 cells were incubated with apolyclonal anti-FcRγ antibody and protein A-sepharose.Immunoprecipitated proteins were separated by SDS-PAGE and blotted onPVDF. Membranes were incubated with a mixture of anti-FcRγ and anti-GPVIantibodies revealed with peroxidase-coupled protein A andchemiluminescence.

FIG. 18: Inhibition of Cvx- or collagen-induced platelet activation byrecombinant human soluble GPVI:Fc.

FIG. 18A: tracing a: platelets were activated by 100 pM Cvx; tracing b:platelet suspension was incubated with 1 μg recombinant human solubleGPVI:Fc for two minutes before the addition of Cvx; tracing c and d:collagen was preincubated with 0.25 μg and 0.5 μg recombinant humansoluble GPVI:Fc for two minutes respectively before addition toplatelets.

FIG. 18B: tracing a: platelets were activated by collagen type I;tracing b: platelets were preincubated with 5 μg of recombinant solubleGPVI:Fc for two minutes before the addition of collagen; tracings c toe: collagen was preincubated with respectively 1 μg, 2.5 μg and 5 μg ofrecombinant soluble GPVI:Fc for two minutes before addition toplatelets. ¹⁴C 5-HT labeled washed platelets were used. The percentageof ¹⁴C 5-HT release measured in each condition is indicated.

FIG. 19: Bleeding time of mice transplanted with bone marrow cellsexpressing GPVI. Irradiated mice transplanted with bone marrow cellsexpressing full length GPVI, the extracellular domain of GPVI, or acontrol were analyzed two months post-transplantation for the recoverytime from a small tail vein incision.

DETAILED DESCRIPTION OF THE INVENTION

The TANGO 268 proteins and nucleic acid molecules comprise a family ofmolecules having certain conserved structural and functional features.As used herein, the term “family” is intended to mean two or moreproteins or nucleic acid molecules having a common structural domain andhaving sufficient amino acid or nucleotide sequence identity as definedherein. Family members can be from either the same or different species.For example, a family can comprises two or more proteins of humanorigin, or can comprise one or more proteins of human origin and one ormore of non-human origin. Members of the same family may also havecommon structural domains.

For example, TANGO 268 proteins of the invention have signal sequences.As used herein, a “signal sequence” includes a peptide of at least about15 or 20 amino acid residues in length which occurs at the N-terminus ofsecretory and membrane-bound proteins and which contains at least about70% hydrophobic amino acid residues such as alanine, leucine,isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. Ina preferred embodiment, a signal sequence contains at least about 10 to40 amino acid residues, preferably about 19-34 amino acid residues, andhas at least about 60-80%, more preferably 65-75%, and more preferablyat least about 70% hydrophobic residues. A signal sequence serves todirect a protein containing such a sequence to a lipid bilayer. Thus, inone embodiment, a TANGO 268 protein contains a signal sequence at aboutamino acids 1 to 20 of SEQ ID NO:3 (SEQ ID NO:4) or at about amino acids1 to 21 of SEQ ID NO:16 (SEQ ID NO:17). The signal sequence is cleavedduring processing of the mature protein.

A TANGO 268 family member consists of one or more of the followingdomains: (1) an extracellular domain; (2) a transmembrane domain; and(3) a cytoplasmic domain. In one embodiment, a TANGO 268 proteincontains an extracellular domain at about amino acid residues 21 to 269of SEQ ID NO:3 (SEQ ID NO:9), a transmembrane domain at about amino acidresidues 270 to 288 of SEQ ID NO:3 (SEQ ID NO:8), and a cytoplasmicdomain at about amino acid residues 289 to 339 of SEQ ID NO:3 (SEQ IDNO:10). In this embodiment, the mature TANGO 268 protein corresponds toamino acids 21 to 339 of SEQ ID NO:3 (SEQ ID NO:5). In anotherembodiment, a TANGO 268 family contains an extracellular domain at aboutamino acid residues 22 to 267 of SEQ ID NO:16 (SEQ ID NO:19), atransmembrane domain at about amino acid residues 268 to 286 of SEQ IDNO:16 (SEQ ID NO:20), and a cytoplasmic domain at about amino acidresidues 287 to 313 of SEQ ID NO:16 (SEQ ID NO:21). In this embodiment,the mature TANGO 268 protein corresponds to amino acids 22 to 313 of SEQID NO:16 (SEQ ID NO:18).

A TANGO 268 family member contains a charged residue, such as arginine,lysine, histidine, glutamic acid, and aspartic acid, in itstransmembrane domain. In one embodiment, a TANGO 268 protein contains acharged amino acid residue, preferably arginine, at amino acid 272 ofSEQ ID NO:3. In another embodiment, a TANGO 268 protein contains acharged amino acid residue, preferably arginine, at amino acid 270 ofSEQ ID NO:16.

A TANGO 268 family member includes a signal sequence. In certainembodiment, a TANGO 268 family member has the amino acid sequence of SEQID NO:3, and the signal sequence is located at amino acids 1 to 18, 1 to19, 1 to 20, 1 to 21 or 1 to 22. In an another embodiment, a TANGO 268family member has the amino acid sequence of SEQ ID NO:16, and thesignal sequence is located at amino acids 1 to 19, 1 to 20, 1 to 21, 1to 22 or 1 to 23. In such embodiments of the invention, theextracellular domain and the mature protein resulting from cleavage ofsuch signal peptides are also included herein. For example, the cleavageof a signal sequence consisting of amino acids 1 to 19 results in anextracellular domain consisting of amino acids 20 to 269 of SEQ ID NO:3and the mature TANGO 268 protein corresponding to amino 20 to 339.

An Ig domain typically has the following consensus sequence, beginningabout 1 to 15 amino acid residues, more preferably about 3 to 10 aminoacid residues, and most preferably about 5 amino acid residues from theC-terminal end of a protein: (FY)-Xaa-C-Xaa-(VA)-COO—, wherein (FY) iseither a phenylalanine or a tyrosine residue (preferably tyrosine),where “Xaa” is any amino acid, C is a cysteine residue, (VA) is eithervaline or an alanine residue (preferably alanine), and COO— is theprotein C-terminus. An Ig-like domain as described herein has thefollowing consensus sequence, beginning about 1 to 15 amino acidresidues, more preferably about 3 to 10 amino acid residues, and mostpreferably about 5 amino acid residues from the domain C-terminus:(FY)-Xaa-C, wherein (FY) is either a phenylalanine or a tyrosine residue(preferably tyrosine), where “Xaa” is any amino acid, and C is acysteine residue. In one embodiment, a TANGO 268 family member includesone or more Ig-like domains having an amino acid sequence that is atleast about 55%, preferably at least about 65%, more preferably at least75%, yet more preferably at least about 85%, and most preferably atleast about 95% identical to amino acids 48 to 88 and/or amino acids 134to 180 of SEQ ID NO:3, which are the Ig-like domains of human TANGO 268(these Ig-like domains are also represented as SEQ ID NO:6 and 7,respectively).

In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least about55%, preferably at least about 65%, more preferably at least about 75%,yet more preferably at least about 85%, and most preferably at leastabout 95% identical to amino acids 48 to 88 and/or amino acids 134 to180 of SEQ ID NO:3, which are the Ig-like domains of human TANGO 268(these Ig-like domains are also represented as SEQ ID NO:6 and 7,respectively), includes a conserved cysteine residue about 8 residuesdownstream from the N-terminus of the Ig-like domain, and has one ormore Ig-like domain consensus sequences as described herein.

In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 48 to 88 and/or amino acids 134 to 180 ofSEQ ID NO:3, which are the Ig-like domains of human TANGO 268 (SEQ IDNO:6 and 7, respectively), includes a conserved cysteine residue 8residues downstream from the N-terminus of the Ig-like domain, has oneor more Ig-like domain consensus sequences as described herein, and hasa conserved cysteine within the consensus sequence that forms adisulfide with said first conserved cysteine.

In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 48 to 88 and/or amino acids 134 to 180 ofSEQ ID NO:3, which are the Ig-like domains of human TANGO 268 (SEQ iIDNO:6 and 7, respectively), includes a conserved cysteine residue 8residues downstream from the N-terminus of the Ig-like domain, has oneor more Ig-like domain consensus sequences as described herein, and hasa conserved cysteine within the consensus sequence that forms adisulfide with said first conserved cysteine.

In yet another embodiment, a TANGO 268 family member includes one ormore Ig-like domains having an amino acid sequence that is at least 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 48 to 88 and/or amino acids 134 to 180 ofSEQ ID NO:3 (SEQ ID NO:6 and 7, respectively), includes a conservedcysteine residue 8 residues downstream from the N-terminus of theIg-like domain, has one or more Ig-like domain consensus sequencesdescribed herein, has a conserved cysteine within the consensus sequencethat forms a disulfide with said first conserved cysteine, and has atleast one TANGO 268 biological activity as described herein.

In another embodiment, the Ig-like domain of TANGO 268 is an Ig domain,which has the following consensus sequence at the C-terminus of thedomain: (FY)-Xaa-C-Xaa-(VA)-COO—, wherein (FY) is either a phenylalanineor a tyrosine residue (preferably tyrosine), where “Xaa” is any aminoacid, C is a cysteine residue, (VA) is a valine or alanine residue, andCOO— is the C-terminus of the domain. In this embodiment, a TANGO 268family member includes one or more Ig-like domains having an amino acidsequence that is at least about 55%, preferably at least about 65%, morepreferably at least 75%, yet more preferably at least about 85%, andmost preferably at least about 95% identical to amino acids 48 to 90and/or amino acids 134 to 182 of SEQ ID NO:3.

In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least about55%, preferably at least about 65%, more preferably at least 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 49 to 89 and/or amino acids 135 to 181 ofSEQ ID NO:16, which are the Ig-like domains of mouse TANGO 268 (theseIg-like domains are also represented SEQ ID NO:22 and 23, respectively).In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least about55%, preferably at least about 65%, more preferably at least about 75%,yet more preferably at least about 85%, and most preferably at leastabout 95% identical to amino acids 49 to 89 and/or amino acids 135 to181 of SEQ ID NO:16 (SEQ ID NO:22 and 23, respectively), includes aconserved cysteine residue about 8 residues downstream from theN-terminus of the Ig-like domain, and has one or more Ig-like domainconsensus sequences as described herein.

In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 49 to 89 and/or amino acids 135 to 181 ofSEQ ID NO:16 (SEQ ID NO:22 and 23, respectively), includes a conservedcysteine residue 8 residues downstream from the N-terminus of theIg-like domain, has one or more Ig-like domain consensus sequences asdescribed herein, and has a conserved cysteine within the consensussequence that forms. a disulfide with said first conserved cysteine.

In another embodiment, a TANGO 268 family member includes one or moreIg-like domains having an amino acid sequence that is at least 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 49 to 89 and/or amino acids 135 to 181 ofSEQ ID NO:16 (SEQ ID NO:22 and 23, respectively), includes a conservedcysteine residue 8 residues downstream from the N-terminus of theIg-like domain, has one or more Ig-like domain consensus sequences asdescribed herein, and has a conserved cysteine within the consensussequence that forms a disulfide with said first conserved cysteine.

In yet another embodiment, a TANGO 268 family member includes one ormore Ig-like domains having an amino acid sequence that is at least 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 49 to 89 and/or amino acids 135 to 181 ofSEQ ID NO:16 (SEQ ID NO:22 and 23, respectively), includes a conservedcysteine residue 8 residues downstream from the N-terminus of theIg-like domain, has one or more Ig-like domain consensus sequences asdescribed herein, has a conserved cysteine within the consensus sequencethat forms a disulfide with said first conserved cysteine, and has atleast one TANGO 268 biological activity as described herein.

In another embodiment, the Ig-like domain of TANGO 268 is an Ig domain,which has the following consensus sequence at the C-terminus end of thedomain: (FY)-Xaa-C-Xaa-(VA)-COO—, wherein (FY) is either a phenylalanineor a tyrosine residue (preferably tyrosine), where “Xaa” is any aminoacid, C is a cysteine residue, (VA) is a valine or alanine residue, andCOO— is the C-terminus of the domain. In this embodiment, a TANGO 268family member includes one or more Ig-like domains having an amino acidsequence that is at least about 55%, preferably at least about 65%, morepreferably at least 75%, yet more preferably at least about 85%, andmost preferably at least about 95% identical to amino acids 49 to 91and/or amino acids 135 to 183 of SEQ ID NO:16, which are the Ig-likedomains of mouse TANGO 268.

In a preferred embodiment, a TANGO 268 family member has the amino acidsequence of SEQ ID NO:6, wherein the aforementioned Ig-like domainconserved residues are located as follows: the N-terminal conservedcysteine residue is located at amino acid residue position 48 (withinthe Ig-like domain SEQ ID NO:3) and the C-terminal conserved cysteineresidue is located at amino acid position 88 (within the Ig-like domainSEQ ID NO:3). In another preferred embodiment, a TANGO 268 family memberhas the amino acid sequence of SEQ ID NO:6, wherein the aforementionedIg-like domain conserved residues are located as follows: the N-terminalconserved cysteine residue is located at amino acid residue position 135(within the Ig-like domain SEQ ID NO:3) and the C-terminal conservedcysteine residue is located at amino acid position 180 (within theIg-like domain SEQ ID NO:3). In another preferred embodiment, a TANGO268 family member has the amino acid sequence of SEQ ID NO:22, whereinthe aforementioned Ig-like domain conserved residues are located asfollows: the N-terminal conserved cysteine residue is located at aminoacid residue position 49 (within the Ig-like domain of SEQ ID NO:16) andthe C-terminal conserved cysteine residue is located at amino acidposition 89 (within the Ig-like domain of SEQ ID NO:16). In anotherpreferred embodiment, a TANGO 268 family member has the amino acidsequence of SEQ ID NO:23, wherein the aforementioned Ig-like domainconserved residues are located as follows: the N-terminal conservedcysteine residue is located at amino acid residue position 135 (withinthe Ig-like domain of SEQ ID NO:16) and the C-terminal conservedcysteine residue is located at amino acid position 181 (within theIg-like domain of SEQ ID NO:16).

Various features of human and mouse TANGO 268 are summarized below.

Human TANGO 268

A cDNA encoding human TANGO 268 was identified by analyzing thesequences of clones present in a human megakaryocyte cDNA library. Thisanalysis led to the identification of a clone, jthealOSeO2, encodingfull-length human TANGO 268. The human TANGO 268 cDNA of this clone is2047 nucleotides long (FIG. 1; SEQ ID NO:1). The open reading frame ofthis cDNA, nucleotides 36 to 1052 of SEQ ID NO:1 (SEQ ID NO:2), encodesa 339 amino acid transmembrane protein (FIG. 1; SEQ ID NO:3) that, asdiscussed below, represents a platelet-expressed collagen receptorglycoprotein.

The signal peptide prediction program SIGNALP (Nielsen, et al., 1997,Protein Engineering 10:1-6) predicted that human TANGO 268 includes an20 amino acid signal peptide (amino acid 1 to about amino acid 20 of SEQID NO:3; SEQ ID NO:4) preceding the mature human TANGO 268 protein(corresponding to about amino acid 21 to amino acid 339 of SEQ ID NO:3;SEQ ID NO:5). The molecular weight of human TANGO 268 withoutpost-translational modifications is 36.9 kDa prior to the cleavage ofthe signal peptide, 34.9 kDa after cleavage of the signal peptide.

Human TANGO 268 is a transmembrane protein that is a collagen receptorexpressed on platelets which consists of one or more of the followingdomains: (1) an extracellular domain; (2) a transmembrane domain; and(3) a cytoplasmic domain. The human TANGO 268 protein contains anextracellular domain at amino acid residues 21 to 269 of SEQ ID NO:3(SEQ ID NO:9), a transmembrane domain at amino acid residues 270 to 288of SEQ ID NO:3 (SEQ ID NO:8), and a cytoplasmic domain at amino acidresidues 289 to 339 of SEQ ID NO:3 (SEQ ID NO:10).

FIG. 2 depicts a hydropathy plot of human TANGO 268. Relativelyhydrophobic regions of the protein are shown above the horizontal line,and relatively hydrophilic regions of the protein are below thehorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence (amino acids 1 to 20 of SEQ ID NO:3; SEQ ID NO:4) on theleft from the mature protein (amino acids 21 to 339 of SEQ ID NO:3; SEQID NO:5) on the right.

Human TANGO 268 comprises two immunoglobulin-like domain sequences atamino acids 48 to 88 and at amino acids 134 to 180 of SEQ ID NO:3; SEQID NO:6 and SEQ ID NO:7. A single N-glycosylation site having thesequence NGSL is present at about amino acids 92 to 95 of SEQ ID NO:3.Nine protein kinase C phosphorylation sites are present in human TANGO268. The first has the sequence TLR (at amino acids 45 to 47 of SEQ IDNO:3), the second has the sequence SSR (at amino acids 64 to 66 of SEQID NO:3), the third has the sequence TYR (at amino acids 177 to 179 ofSEQ ID NO:3), the fourth has the sequence SSR (at amino acids 184 to 186of SEQ ID NO:3), the fifth has the sequence TNK (at amino acids 235 to237 of SEQ ID NO:3), the sixth has the sequence TSR (at amino acids 243to 245 of SEQ ID NO:3), the seventh has the sequence SPK (at amino acids250 to 252 of SEQ ID NO:3), the eighth has the sequence SRR (at aminoacids 293 to 295 of SEQ ID NO:3), and the ninth has the sequence TRK (atamino acids 318 to 320 of SEQ ID NO:3). Four casein kinase IIphosphorylation sites are present in human TANGO 268. The first has thesequence SGGD (at amino acids 126 to 129 of SEQ ID NO:3), the second hasthe sequence SSRD (at amino acids 184 to 187 of SEQ ID NO:3), the thirdhas the sequence SVAE (at amino acids 219 to 222 of SEQ ID NO:3), andthe fourth has the sequence SPKE (at amino acids 250 to 253 of SEQ IDNO:3). Human TANGO 268 has two tyrosine kinase phosphorylation siteshaving the sequences KEGDPAPY (at amino acids 147 to 154 of SEQ ID NO:3)and KNPERWY (at amino acids 155 to 161 of SEQ ID NO:3). Human TANGO 268has five N-myristylation sites. The first has the sequence GLCLGR (atamino acids 12 to 17 of SEQ ID NO:3), the second has the sequence GSLWSL(at amino acids 93 to 98 of SEQ ID NO:3), the third has the sequenceGGDVTL (at amino acids 127 to 132 of SEQ ID NO:3), the fourth has thesequence GTYRCY (at amino acids 176 to 181 of SEQ ID NO:3), and thefifth has the sequence GGQDGG (at amino acids 323 to 328 of SEQ IDNO:3). Human TANGO 268 is likely to be involved in cell signaling viainteraction with a second receptor component. A charged residue ispresent in the transmembrane domain of human TANGO 268 (arginine atamino acid 272 of SEQ ID NO:3), which is a hallmark of platelet collagenreceptors, and which can function as an interaction site for associationwith other membrane proteins, a second receptor component, e.g., FcRγ.

FIG. 5A depicts the alignment between the first immunoglobulin-likedomain of human TANGO 268 (from amino acid residues 41 to 90 of SEQ IDNO:3) and a typical immunoglobulin domain (SEQ ID NO:13; AccessionNumber PF00047). FIG. 5B depicts the alignment between the secondimmunoglobulin-like domain of human TANGO 268 (from amino acid residues127 to 182 of SEQ ID NO:3) and a typical immunoglobulin domain (SEQ IDNO:13; Accession Number PF00047).

Northern blot analysis of human TANGO 268 expression demonstratesexpression in bone marrow, fetal liver, and peripheral blood leukocytes.Fetal liver expression reveals one human TANGO 268 mRNA band that isapproximately 2 kb. Human TANGO 268 expression was not detected in thefollowing tissues: spleen, lymph node, thymus, brain, heart, skeletalmuscle, colon, kidney, liver, small intestine, placenta, or lung.Further analysis predicts that TANGO 268 is specific to themegakaryocyte lineage of hematopoietic cells.

Clone EpthEa11d1, which encodes human TANGO 268, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on March 30, 1999 and assigned Accession Number 207180.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

FIG. 3 shows an alignment of the human TANGO 268 coding region (SEQ IDNO:2) with the human monocyte inhibitory receptor precursor proteincoding region (SEQ ID NO:24). The human monocyte inhibitory receptor hasbeen shown to downregulate activation responses by phosphatases. Thenucleotide sequences of coding regions of human monocyte inhibitoryreceptor precursor and human TANGO 268 are 37.7% identical. Thefull-length nucleic acid sequence of human TANGO 268 (SEQ ID NO:1)exhibits 49.9% identity to the full-length nucleic acid human monocyteinhibitory receptor precursor (SEQ ID NO:11; Accession Number U91928).

FIG. 4 shows that there is an overall 23% identity between the aminoacid sequence of the human TANGO 268 protein and the amino acid sequenceof the human monocyte inhibitory receptor protein (SEQ ID NO:12;Accession Number U91928).

In general, human TANGO 268 has most homology to various members of theimmunoglobulin superfamily that include NK inhibitory and activatingreceptors and Fc receptors. Specifically, TANGO 268 represents aplatelet-specific collagen receptor previously described as GlycoproteinVI (GPVI), and thus can be involved in hemostasis and thrombosis. Thefact that TANGO 268 represents GPVI was suggested by the following: (1)TANGO 268 and GPVI are both preferentially expressed in themegakaryocytic cells; (2) the molecular mass of the 40 kDaunglycosylated TANGO 268 is predicted to be approximately 62 kDa, theapparent molecular mass of GPVI, upon N— and O-linked glycosylation; (3)the presence of two immunoglobulin-like domains in TANGO 268 indicatesthat like GPVI, TANGO 268 interacts with the FcRγ; (4) the absence of alarge intracytoplasmic tail, suggesting that this membrane-boundglycoprotein has no signaling role but associates with another member ofthe Ig family (e.g., FcRγ) protein to transduce a signal; and (5) thepresence of a charged residue (arginine) in the transmembrane domain ofTANGO 268 which is predicted to be present in GPVI based on itsassociation with the FcRγ. Experimental data confirming that TANGO 268does, indeed, represent GPVI are presented below.

Mouse TANGO 268

A cDNA encoding mouse TANGO 268 was identified by analyzing thesequences of clones present in a mouse megakaryocyte cDNA library. Thisanalysis led to the identification of a clone, jtmea105e02, encodingfull-length mouse TANGO 268. The murine TANGO 268 cDNA of this clone is1163 nucleotides long (FIG. 6; SEQ ID NO:14). The open reading frame ofthis cDNA, nucleotides 63 to 1001 of SEQ ID NO:14 (SEQ ID NO:15),encodes a 313 amino acid transmembrane protein (FIG. 6; SEQ ID NO:16).

The signal peptide prediction program SIGNALP (Nielsen, et al., 1997,Protein Engineering 10:1-6) predicted that mouse TANGO 268 includes a21amino acid signal peptide (amino acid 1 to amino acid 21 of SEQ IDNO:16)(SEQ ID NO:17) preceding the mature mouse TANGO 268 protein(corresponding to amino acid 22 to amino acid 313 of SEQ ID NO:16)(SEQID NO:18). The molecular weight of mouse TANGO 268 withoutpost-translational modifications is 34.5 kDa prior to the cleavage ofthe signal peptide, 32.3 kDa after cleavage of the signal peptide.

Mouse TANGO 268 is a transmembrane protein which consists of one or moreof the following domains: (1) an extracellular domain; (2) atransmembrane domain; and (3) a cytoplasmic domain. The mouse TANGO 268protein contains an extracellular domain at amino acid residues 1 to 267of SEQ ID NO:16 (SEQ ID NO:19), a transmembrane domain at amino acidresidues 268 to 286 of SEQ ID NO:16 (SEQ ID NO:20), and a cytoplasmicdomain at amino acid residues 287 to 313 of SEQ ID NO:16 (SEQ ID NO:21).

FIG. 7 depicts a hydropathy plot of mouse TANGO 268. Relativelyhydrophobic regions of the protein are shown above the horizontal line,and relatively hydrophilic regions of the protein are below thehorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence on the left from the mature protein on the right.

Mouse TANGO 268 comprises two immunoglobulin-like domain sequences atamino acids 49 to 89 and at amino acids 135 to 181 of SEQ ID NO:16; SEQID NO:22 and SEQ ID NO:23. Two N-glycosylation sites having thesequences NGSH and NITA are present in mouse TANGO 268 at amino acids 93to 96 and at amino acids 244 to 247 of SEQ ID NO:16, respectively. Sixprotein kinase C phosphorylation sites are present in mouse TANGO 268.The first has the sequence TLK (at amino acids 132 to 134 of SEQ IDNO:16), the second has the sequence TYR (at amino acids 178 to 180 ofSEQ ID NO:16), the third has the sequence SSR (at amino acids 224 to 226of SEQ ID NO:16), the fourth has the sequence TNK (at amino acids 233 to235 of SEQ ID NO:16), the fifth has the sequence TEK (at amino acids 239to 241 of SEQ ID NO:16), and the sixth has the sequence SRK (at aminoacids 291 to 293 of SEQ ID NO:16). Two casein kinase II phosphorylationsites are present in mouse TANGO 268. The first has the sequence SFDE(at amino acids 140 to 143 of SEQ ID NO:16), and the second has thesequence STIE (at amino acids 237 to 240 of SEQ ID NO:16). Mouse TANGO268 has two tyrosine kinase phosphorylation sites having the sequencesKEGDTGPY (at amino acids 148 to 155 of SEQ ID NO:16) and KRPEKWY (atamino acids 156 to 162 of SEQ ID NO:16). Mouse TANGO 268 has twoN-myristylation sites. The first has the sequence GSHWSL (at amino acids94 to 99 of SEQ ID NO:16), and the second has the sequence GTYRCY (atamino acids 177 to 182 of SEQ ID NO:16). A c-AMP- and c-GMP-dependentprotein kinase phosphorylation site is present in the mouse TANGO 268having the sequence RRPS (at amino acids 226 to 229 of SEQ ID NO:16). AnABC transporter family signature is present in mouse TANGO 268 havingthe sequence YAKGNLVRICLGATI (at amino acid residues 263 to 277 of SEQID NO:16). Mouse TANGO 268 does not include any conspicuous inhibitoryor activation motifs in the cytoplasmic domain. Mouse TANGO 268 may beinvolved in cell signaling via interaction with a second receptorcomponent. A charged residue is present in the transmembrane domain ofmouse TANGO 268 (arginine at amino acid 270 of SEQ ID NO:16), which mayfunction as an interaction site for association with other membraneproteins, a second receptor component, e.g., FcRγ.

FIG. 10A depicts the alignment between the first immunoglobulin-likedomain of mouse TANGO 268 (from amino acid residues 42 to 91 of SEQ IDNO:16) and a typical immunoglobulin domain (SEQ ID NO:13; Accession No.PF00047). FIG. 10B depicts the alignment between the secondimmunoglobulin-like domain of mouse TANGO 268 (from amino acid residues128 to 183 of SEQ ID NO:16) and a typical immunoglobulin domain (SEQ IDNO:13; Accession No. PF00047).

In situ expression experiments with a TANGO 268 anti-sense probe(nucleotides 69 to 670 of SEQ ID NO:14) reveal that during embryogenesismouse TANGO 268 is expressed exclusively in the liver. The signalpattern is strong and multifocal, suggestive of expression by ascattered cell population. In adult tissues, expression of TANGO 268 inliver is no longer observed but a strong, multifocal signal is seen inspleen. The number of multifocal signals observed in the spleen issignificantly reduced compared to the number observed in embryonicliver. All other adult tissues tested negative for TANGO 268 (i.e., nosignal was observed in the brain, eye, harderian gland, submandibulargland, bladder, white fat, stomach, brown fat, heart, adrenal gland,colon, small intestine, liver, placenta, thymus, lymph node, spleen,lung, spinal cord, pancreas, skeletal muscle or testes). A sense probeanalogous to the anti-sense TANGO 268 probe tested on the same tissuesyielded no signal.

The signal pattern and restricted tissue expression observed duringembryogenesis and in adult tissues was identical to that seen with aprobe for TANGO 69, a gene known to be expressed by megakaryocytes (PCTPublication Number WO 99/11662, published on Mar. 11, 1999). Like TANGO69, TANGO 268 was also cloned from a megakaryocyte library. These data,therefore, indicate that TANGO 268 is expressed by megakaryocytes duringembryogenesis and in adult mice.

In general, mouse TANGO 268 has most homology to various members of theimmunoglobulin superfamily that includes NK inhibitory and activatingreceptors and Fc receptors. The full-length nucleic acid sequence ofmouse TANGO 268 exhibits 35.6% identity to the full-length nucleic acidhuman monocyte inhibitory receptor precursor (SEQ ID NO:11; AccessionNumber U91928). FIG. 8 shows an alignment of the mouse TANGO 268 codingregion (SEQ ID NO:15) with the human monocyte inhibitory receptorprecursor protein coding region (SEQ ID NO:24). The nucleotide sequencesof the coding regions of human monocyte inhibitory receptor precursorand mouse TANGO 268 are 34.4% identical. The nucleotide sequences of thefull-length human monocyte inhibitory receptor precursor (SEQ ID NO:11;Accession Number U91928) and full-length mouse TANGO 268 (SEQ ID NO:14)are 35.6% identical. FIG. 9 shows that there is an overall 20.3%identity between the mouse TANGO 268 amino acid sequence and the humanmonocyte inhibitory receptor protein amino acid sequence (SEQ ID NO:12;Accession Number U91928).

FIG. 11 shows that there is an overall 64.4% identity between theprecursor human TANGO 268 amino acid sequence (SEQ ID NO:3) and theprecursor mouse TANGO 268 amino acid sequence (SEQ ID NO:16). Thishomology is spread throughout the molecule, but is slightly higher (78%)over the immunoglobulin-like domains Interestingly, both human and mouseGPVI contain conserved variants of the WSXWS box (residues 96-100 and192-196). This motif is a signature of class I hematopoietic receptorsbut variants are also found in the sequences of all Killer-cellInhibitory receptors (KIR) (Fan et al., 1997, Nature 389: 96-100). Thesemotifs have been shown to contribute to tertiary folding. GPVI has arelatively short cytoplasmic tail with no obvious signaling motifsanalogous to the ITAM's and immunoreceptor tyrosine-based inhibitorymotifs (ITIM's) of other signaling receptors. However, GPVI present apositively charged residue in the transmembrane domain allowing it toform complexes with the FcRγ chain which acts as signaling subunit(Poole et al., 1997, cited below).

Clone EpTm268, which encodes mouse TANGO 268, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on Jun. 14, 1999 and assigned Accession No. PTA-225.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

Functional and Structural Analyses Demonstrating that TANGO 268 isGlycoprotein VI

Described below are both functional (ligand binding, cell adhesion andplatelet activation) and structural (immunoblot and tissue expression)analysis demonstrating that TANGO 268 is glycoprotein VI.

A. Ligand Binding Assay

Convulxin (Cvx) is a protein purified from the venom of Crotallusdurissus terrificus. Cvx is known to act as a potent platelet agonist,and has been shown to bind specifically to GPVI. Described below are Cvxligand binding studies demonstrating that TANGO 268 specifically bindsCvx.

The ligand binding assay was performed as follows: approximately 5×10⁹human platelets per milliliter, and 10⁶ expression vectoronly-transfected CHO cells and full-length TANGO 268 containingexpression vector-transfected CHO cells were lysed for 30 min at 4° C.in lysis buffer comprising 10 mM Tris, 100 mM NaCl, 5 mM EDTA, pH 8containing 0.1% Nonidet P40, 2 mM PMSF, 5KIU aprotinin and 20 μMleupeptin (Jandrot-Perrus et al., 1997, J. Biol. Chem. 272:27035-27041;Lagrue et al, 1999, FEBS Lett. 448(1):95-100). Approximately 8 μg ofplatelet lysate and 40 to 80 μg of CHO cell lysates (expression vectoronly-transfected and TANGO 268-transfected) were separated on 10%acrylamide slab gels (miniprotean II Biorad) in the presence of SDS andthen transferred to a PVDF membrane (Amersham). The membrane wassaturated with 5% (w/v) non-fat dry milk in PBS. Ligand blotting wasperformed by the incubating membrane in the presence of ¹²⁵I-Cvx (3×10⁵cpm/ml) in PBS pH 7.4 containing 0.5% (w/v) dry milk and 0.2% Tween 20for 4 hours.

The Cvx utilized in the ligand binding assay was purified from the venomof Crotallus durissus terrificus by two successive chromatography steps(Francischetti et al., 1997, Toxicon 35:121728) and radiolabeled.Briefly, lyophilized venom from Crotallus durissus terrificus wassolubilized in ammonium formate 0.1M, NaCl 0.3 M, pH 3.1 and proteinswere separated on a G75 column equilibrated in the same buffer. Cvxcontained in the first eluted peak, as assessed by gel electrophoresisand platelet activating activity, was lyophilized. Second, Cvx wassolubilized in Tris 0.1M pH8.5 containing 6 M urea (Tris urea buffer)and further purified by chromatography on a G100 column equilibrated inthe same buffer. Fractions containing purified Cvx were pooled, dialyzedand lyophilized. After solubilization in the Tris urea buffer, Cvx wasdialyzed against PBS (20 mM phosphate, 150 mM NaCl, pH 7.4). Cvx (100μg) was radiolabeled with 0.5 mCi Na¹²⁵I (Amersham) using lodogen(Pierce Chemical Corp.) according to published procedure (Jandrot-Perruset al., 1997, J. Biol. Chem. 272:27035-27041). lodinated Cvx wasseparated from free ¹²⁵I by gel filtration on a G25 sephadex column(Pharmacia) in PBS. The activity of ¹²⁵I-Cvx was tested on humanplatelet aggregation.

Following the incubation of the membrane with ¹²⁵I-Cvx, the membrane waswashed and ligand binding was detected by autoradiography on X-Omat MAfilms (Kodak). Ligand blotting with ¹²⁵I-Cvx (FIG. 12) revealed onespecific band in platelet lysates at 56-58 kDa (lane 1), whichrepresents a band previously identified as GPVI (Jandrot-Perrus et al.,1997, J. Biol. Chem. 272:27035-27041). No positive band was observed inlysates (60 μg) from control expression vector only-transfected cells(lane 2). In lysates from CHO cells transfected with TANGO 268expression vector (60 μg), a positive band migrating at 52-54 kDa wasclearly observed (lane 3).

The ligand binding studies demonstrate that convulxin binds to amolecule present on TANGO 268 transfected cells (and not on vector onlytransfected cells), which has a molecular weight very similar to themolecular weight of GPVI (FIG. 12). The small apparent difference insize between the band in platelet lysates and in CHO lysates can beaccounted for by cell-type specific discrepancies in proteinglycosylation.

This result demonstrates that convulxin binds to TANGO 268 and thatTANGO 268 has a similar or identical molecular weight as GPVI. SinceGPVI is the platelet receptor for Cvx (Jandrot-Perrus et al., 1997,Journal of Biological Chemistry 272:27035-27041) and TANGO 268 ispreferentially expressed in megakaryocytes, this functional evidenceindicates that TANGO 268 is GPVI.

B. Immunoblotting Assay

Structural evidence is presented herein that further supports TANGO 268as corresponding to GPVI. In particular, the immunoblotting resultspresented herein demonstrate that an IgG preparation containingantibodies directed against GPVI binds specifically to TANGO 268polypeptide. These studies further demonstrate that binding issuccessfully competed away when Cvx is introduced.

The immunoblotting assay was performed as follows: platelet, expressionvector-only transfected and TANGO 268 containing expressionvector-transfected CHO cell lysates were generated as described in A.above. Approximately 8 μg of platelet lysate and 40 to 80 μg of CHO celllysate (either expression vector-only transfected or TANGO268-transfected) were separated on 10% acrylamide slab gels (miniproteanII Biorad) in the presence of SDS and then transferred to a PVDFmembrane (Amersham). The membrane was saturated with 5% (w/v) non-fatdry milk in PBS, and then incubated for 2 hours at room temperature with9 μg/ml anti-GPVI IgG in PBS, pH 8.6 containing 0.02% (v/v) Tween 20.

Alternatively, for the competition assay, the membrane was incubated for2 hours at room temperature with 9 μg/ml anti-GPVI IgG in PBS, pH 8.6containing 0.02% (v/v) Tween 20 in the presence of a high concentrationof cold Cvx (0.5 μM).

The IgG preparation utilized in this assay was generated by purifyingIgG from serum of a patient exhibiting idiopathic thrombocytopenicpurpura (ITP) (Sugiyama et al., 1987, Blood 69: 1712-1720) as describedin Jandrot-Perrus et al., 1997, J. Biol. Chem. 272:27035-27041.Following the incubation with the antibody composition, the membrane waswashed and incubated with peroxidase-coupled protein A (Amersham) for 2hours at room temperature. The immunoblots were developed using enhancedchemiluminescence detection (Amersham).

As shown in FIG. 13A, immunoblotting with the IgG revealed a 56-58 kDain platelet lysates (lane 1), which corresponds to the molecular mass ofGPVI. The high molecular weight band detected in platelet lysatescorresponds to platelet IgGs revealed by protein A. The presence of a52-54 kDa band was detected in TANGO 268-transfected CHO cell lysates(FIG. 13A, lane 3) but not in expression vector only-transfected CHOcell lysates (lane 2) demonstrating that TANGO 268 shares epitopesimilarities with GPVI. The low molecular weight bands of moderateintensity observed in FIG. 13A, lanes 2 and 3 are non-specific bandssince they were detected in both control, expression vectoronly-transfected and TANGO 268 transfected CHO cell lysates.

The results from the competition assay performed further demonstrate thesimilarities between TANGO 268 and GPVI. In particular, as shown in FIG.13B, cold 0.5 μM Cvx successfully competes with and inhibits anti-GPVIbinding to GPVI on platelet lysates (lane 1), and likewise, the 52-54kDa band revealed by the anti-GPVI IgG in TANGO 268-tranfected cellslysates (lane 3), was inhibited in the presence of 0.5 μM Cvx.

In summary, the results from both the ligand binding assays andimmunblotting assays described above provide both functional (i.e.,binding of Cvx to TANGO 268) and immunological evidence (i.e.,recognition by anti-GPVI IgG) that TANGO 268 does, indeed, representGPVI polypeptide.

C. Tissue Expression of Tango 268/GPVI

To further study tissue distribution of both mouse and human Tango268/GPVI, Northern blot, RT-PCR and in situ hybridizations wereperformed. The results presented herein confirm and extend theexperimental results presented above.

Materials & Methods

In situ hybridization: In situ hybridization (ISH) was performed withday 12.5 C57BL/B6 mouse embryos and normal 4- to 6-week-old C57BLJ6mouse femurs. Tissues were fixed in 10% formalin, paraffin embedded andsubsequently sectioned at 4 μm onto Superfrost/plus slides. Femurs weredecalcified in TBD-2 (Shandon, Pittsburgh, Pa.) prior to paraffinembedding. Sections were deparaffinized in xylene, hydrated through aseries of graded ethanol washes and placed in DEPC-treatedphosphate-buffered saline (PBS) pH7.4 before being processed for ISH.Sections were incubated in 20 ug/ml proteinase K (Sigma) in DEPC-PBS for15 minutes at 37° C. and then immersed in 4% formaldehyde/PBS for 5minutes. Sections were treated with 0.2N HCl for 10 minutes followed byDEPC-PBS. Sections were rinsed in 0.1M triethanolamine-HCl (TEA, pH8.0), incubated in 0.25% acetic anhydride-TEA for 10 minutes, rinsed inDEPC-PBS, dehydrated through a series of graded ethanol washes and airdried. Labeling and hybridization of ³⁵S-radiolabeled (2.5×10⁷ cpm/ml)cRNA antisense and sense RNA probes encoding a 599 pb fragment of the 5′end of the GPVI gene (generated with the PCR primers forward 5′CAGCCTCACCCACTTTCTTC-3′ (SEQ ID NO:25), nt 8-27 and reverse5′-CCACAAGCACTAGAGGGTCA 3′ (SEQ ID NO:26), nt 607-588) were performed aspreviously described (Busfield et al., 1997, Mol. Cell. Biol. 17:4007-14). Following hybridization, sections were dehydrated rapidlythrough serial ethanol-0.3 M sodium acetate before being air dried,dipped in a nuclear track emulsion (NTB-2: Eastman Kodak, Rochester,N.Y.) and exposed for 60 days at room temperature. Slides were developedwith D-19 (Kodak, Rochester, N.Y.), stained with hematoxylin andeosin-Y, and coverslipped.

Cell lines: The HEL (erythroid/MK), U937 (monoblast), K562 (erythroid),CEM (T cell), HEPG2 and Hela cell lines were obtained from American TypeCulture Collection (ATCC, Manassas, Va.) and the FDC-P1 and 32D cellsfrom D. Metcalf (The Walter and Eliza hall Institute, Melbourne,Australia). The UT7 (erythroid/MK) transduced by c-mpl (Hong et al.,1998, Blood 91:813-822) TF1 (erythroid), KG1 (myeloblast), HL60(myeloblast/promyelocyte), MO-7E (MK), Meg-01 (MK) and DAMI (MK) wereobtained from the different laboratories which derived them (Avanzi etal., 1988, Br. J. Haematol. 69: 359-366) (Collins et al., 1977, Nature270: 347-349) (Greenberg et al., 1988, Blood 72: 1968-1977) (Kitamura etal., 1989, J. Cell Physiol. 140: 323-334) (Koeffler and Golde, 1977,Science 200:1153-1155.) (Komatsu et al., 1991, Cancer Res. 51: 341-348)(Ogura et al., 1985, Blood 66:1364-1392).

HEL, U937 HL60, Meg-01, KGland K562 human cell lines were cultured inIMDM (Gibco/BRL, Grand Island, N.Y.), 10% FCS (Stem cell technology,Vancouver, BC, Canada). The c-mpl UT7, TF1 and MO-7E are factordependent and were grown either in the presence of 2 ng/ml GM-CSF or 10ng/ml PEG-rHuMGDF in IMDM 10% FCS. CEM and Hela were grown in RPMI(Gibco/BRL, Grand Island, N.Y.). FDC-P1, 32D and Ba/F3 murine cell lineswere cultured in DMEM (Gibco/BRL, Grand Island, N.Y.), 10% FCS (Stemcell technology, Vancouver, BC, Canada). Cultures were performed at 37°C. in a fully humidified atmosphere of 5% CO2.

Samples: Human megakaryocytes were obtained as described for the humanlibraries from mobilized or cord blood CD34⁺ cord blood. A fetal liverwas obtained from abortion at 12-week gestation after obtaining informedconsent.

Northern Blot/RT PCR analysis: Human multiple tissue northern blots,purchased from Clontech (Palo Alto, Calif.) were hybridized to a 1.0 kbhuman GPVI probe as described by the manufacturer. Total RNA wasisolated using RNA PLUS (Bioprobe systems, France), a modification ofthe acid-guanidinium thiocyanate-phenol-chloroform extraction method ofChomczynski et Sacchi (Chomczynski and Sacchi 1987). RNA was reversetranscribed with random hexamers using SUPERSCRIPT reverse transcriptase(Gibco BRL/Life Technologies, Cergy Pontoise, France).

For human cell lines and tissues, after reverse transcription, eachsample was subjected to a specific amplification of GP VI and β₂microglobulin cDNA. The sequences of the specific primers were: for GPVI sense primer 5′-TTCTGTCTTGGGCTGTGTCTG-3′ (SEQ ID NO:27) andanti-sense primer 5′-CCCGCCAGGATTATTAGGATC-3′(SEQ ID NO:28), for β₂microglobulin sense primer 5′-CCTGAAGCTGACAGCATTCGG-3′ (SEQ ID NO:29)and anti-sense primer CTCCTAGAGCTACCTGTGGAG-3′ (SEQ ID NO:30). PCR wasperformed in 25 μl reaction mixture containing 0.3 U Taq polymerase(ATGC Noisy-le-Grand, France), 200 μM dNTP, 30 pmol of oligonucleotidesense and 30 pmol of antisense for GP VI amplification and 10 pmol ofoligonucleotide sense and 10 pmol of antisense for β₂ microglobulinamplification, in ATGC buffer. The reaction mixture was subjected todenaturation for 5 min at 95° C. amplified by 35 cycles as follows:denaturation for 30 seconds at 94° C., annealing for 30 seconds at 60°C. and extension at 72° C. for 1 min, with a final 7 min extension at72° C. in a thermocycler 2400 (Perkin Elmer Co, Courtaboeuf, France).PCR products (9 μl) were electrophoresed on a 2% agarose gel. Fragmentswere visualized by illumination after ethidium bromide staining.MassRuler DNA Ladder, low Range (MBI Fermentas, Amherst, N.Y.) is usedas marker.

Results

Human tissues were studied using Northern blot or RT-PCR analysis.Northern blots (FIG. 4D) revealed no specific message in brain, heart,skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine,placenta, lung or lymph nodes. A 2 kb transcript was only observed inbone marrow and fetal liver. A signal was inconsistently observed withperipheral blood cells, probably due to platelet RNA contamination insome samples. Indeed, transcripts for platelet glycoprotein IIb (GPIIb),a platelet specific protein, were also detected in these positivesamples.

Using RT-PCR analysis, no GPVI transcript was detected in blood cellsexcept in platelets. In cell lines, a strong PCR signal was observed inthe HEL, MEG01, DAMI and TPO-stimulated MO7E and mpl transduced UT7 celllines (Hong et al., 1998, Blood 91:813-822). A very low signal was alsodetected in the K562 and KG1 cell lines, two cell lines also expressingGPIIb at low level, but no expression was detected in the HEPG2, CEMT,TF1, U937, HL60 and Hela cells. CD41 positive cells (including more than95% megakaryocytes) isolated from normal cord blood orchemotherapy-induced mobilized peripheral blood displayed a strongRT-PCR signal. Fetal liver cells expressed a moderate level ofexpression compared to megakaryocyte-enriched samples (FIG. 14C)

Mouse tissues were studied using northern blot and ISH analysis. ISHreveals that GPVI was exclusively found in the liver duringembryogenesis (FIG. 14A). The signal pattern was strong and multifocal,suggestive of expression by a scattered cell population. This signal wasobserved at embryonic day 13.5, 14.5, 16.5 and decreased in intensity atday 18.5 and in 1.5 day old new born. In adult, expression in liver wasno longer observed but a strong, multifocal signal was seen in spleenand in the bone marrow. No signal was observed in any other tissuesincluding brain, eye, harderian gland, submandibular gland, bladder,white fat, stomach, brown fat, heart, adrenal gland, colon, smallintestine, liver, placenta, thymus, lymph node, lung, spinal cord,pancreas, skeletal muscle, testes. Photoemulsion processing of thespleen and bone marrow showed that this expression was restricted tomegakaryocytes (FIG. 14B).

In conclusion, despite screening a large number of human and mousetissues, GPVI expression was only detected in megakaryocytes/platelets.This result strongly suggests that GPVI is restricted to thishematopoietic lineage. Presently there are very few molecules that arespecific to the megakaryocyte lineage. GPIIb (integrin αIIβ), which waslong considered to be the prototypic megakaryocyte marker, is alsoexpressed on a subset of hematopoietic progenitors. Other megakaryocyteproteins such as GPIbα, β and GPIX (CD42) are also expressed byactivated endothelial cells. Only PF4 appears to be specific to themegakaryocyte/platelet lineage. For this reason the PF4 promoter hasbeen used to target the megakaryocytes in various transgenic models(Ravid et al., 1991, Proc. Nat'l. Acad. Sci. USA 88:1521-5.). Thus, theGPVI promoter can also be used to target specifically the megakaryocytelineage. For example, the polynucleotides of the invention can be usedto specifically target, via homologous recombination, a gene of interestinto the GPVI locus under the control of the GPVI promoter.Alternatively, the GPVI promoter region can be cloned using standardtechniques known to those in the art (e.g., probing a genomic library byhybridization to the 5′ end of the cDNAs of the invention, and morespecifically, detecting hybridization of the human TANGO 268 clone to ahuman genomic library of chromosome 19 in particular).

D. Flow Cytometry To Study Cell Surface Expression of Tango 268/GPVI

In order to determine whether the recombinant GPVI was expressed at thecell surface, different human or murine hematopoietic cell lines weretransduced with recombinant retroviruses expressing human or murine GPVIand with the control retrovirus.

Materials & Methods

GPVI expressing cell lines: CHO cells were transfected usinglipofectamine (Gibco-BRL, Grand Island, N.Y.), according tomanufacturer's instructions. The expression vector (pMET, MillenniumPharmaceuticals, Cambridge, MA) containing the full length GPVI cDNA,driven by a SRalpha promoter, was isolated from the cDNA library.Control CHO cells were transfected with the empty vector. Cells werecollected 2 days after transfection and lysed in 12 mM Tris, 300 mMNaCl, 12 mM EDTA, containing 2 PM leupeptin, 2 mM PMSF, 5 KIU aprotinin,and 0.2% (v/v) NP40 (Sigma, St. Louis, Mo.). After 20 min at 4° C. underagitation, samples were centrifuged at 13,000 g for 15 min at 4° C. andthe supernatants frozen at −80° C. for analysis.

The human cell lines HEL, U937 and K562 and the murine cell linesFDC-P1, 32D and Ba/F3 murine cell lines were engineered (Burns et al.,1993, Proc. Nat'.l Acad. Sci. USA 90: 8033-7) to express GPVI using thepMSCVpac retrovirus (Hawley et al., 1994, Gene Ther. 1: 136-8). Briefly,viruses carrying the full length cDNA encoding human GPVI or murine GPVIwere constructed using base perfect PCR amplified fragments of the cDNAs(Clontech laboratories Inc, Palo Alto, Calif.). Viral supernatants weregenerated into the 293-EBNA cells (Invitrogen, Carlsbad, Calif.) bytransfecting the retroviral construct and two pN8epsilon vectorscontaining the gag/pol genes from the murine moloney leukemia virus(MMLV) or the Vesicular Stomatitis Virus envelope glycoprotein G (VSV-G)gene. Concentrated viral supernatants were prepared by centrifugation at4° C. using a SW28 rotor at 50,000×g (25,000 rpm) for 2 hr. Pellets wereresuspended in 1.5 ml of DMEM for 24 hr at 4 C, shaken at 4° C. for 24hours and frozen at −80° C. For transduction, cell lines were incubatedwith the viral supernatant over-night in 24 well plates, 10×10⁵ cell/ml,and selected during one week using puromycin (4 μg/ml, Sigma, St. Louis,Mo.). Human and murine GPVI were transduced in human and murine celllines, respectively. Expression of the genes was verified using PCRanalysis. The control cells were transduced with the empty virus.

Convulxin and Bothrojaracin preparation: Convulxin (Cvx) was purifiedfrom the venom of Crotalus durissus terrificus mainly as described byFrancischetti et al, using a two step gel filtration procedure ofsephadex G75 (Pharmacia Biotech, Uppsala, Sweden) followed by sephacrylS100 (Pharmacia Biotech, Uppsala, Sweden). Cvx was labeled with ¹²⁵Iusing the iodogen procedure (Pierce Chemical Co, Rockford, Ill.) andNa¹²⁵I (Amersham, Les Ulis, France). Cvx was coupled to FITC by mixingCvx in 50 mM NaHCO3, 150 mM NaCl, pH 9.5 with a 100 fold molar excess ofFITC (Aldrich, St Quentin Fallavier, France) overnight at 4° C.FITC-coupled Cvx was separated from free FITC by chromatography on asephadex G25 column (PD10 Pharmacia Biotech, Uppsala, Sweden) in 20 mMphosphate, 150 mM NaCl pH 7.4. (PBS). Bothrojaracin, a specific thrombininhibitor purified from the venom of Bothrops jararaca as previouslydescribed (Arocas et al., 1996, Biochemistry 35: 9083-9) was coupled toFITC using the same procedure.

Flow cytometry: Cells transduced with the human or murine GPVI virusesor the control virus were incubated in the presence of 20 nM FITC-Cvx orFITC-bothrojaracin for 60 min. at room temperature. After dilution inPBS cells were analyzed by FACSort flow cytometer (Becton Dickinson,Franklin Lakes, N.J.).

Results

It was observed that the cell lines used for this study expressFcRγ-chain, as indicated by immunoblotting studies using a polyclonalanti-FcRγ antibody. Functional characterization of recombinant GPVI wasperformed using transfected cells which have either no (U937, FDC-P1) orlow (HEL) levels of endogenous GPVI. Unlike DAMI cells which do expressGPVI mRNA, this allowed us to measure responses independent ofendogenous GPVI.

Transduced cells were analyzed by flow cytometry using FITC conjugatedCvx. As a control, we used FITC conjugated bothrojaracin, another snakevenom protein structurally very close to Cvx but a pure thrombininhibitor that does not bind to platelets. Transduction of murine 32Dcells with a retrovirus expressing murine GPVI resulted in a strongCvx-associated staining compared to cells transduced with the controlvirus, indicating that these cells express GPVI at their surface (FIG.15). Similar results were obtained with FDC-P1, and Ba/F3 (all murinecell lines) and with K562 and U937, indicating that murine or human GPVIare expressed at the surface of all these cell lines after transduction.Cvx was found to bind to the wild type HEL cells but the binding wasclearly increased after retroviral transduction indicating an increasedexpression in cells already constitutively expressing GPVI.

Discussion

This ligand binding fluorescence analysis shows that Cvx binds to thehuman recombinant protein in U937 and K562 cells and to the mouserecombinant protein in FDC-P1, 32D and Ba/F3. It is known that Cvxrecognized mouse GPVI from studies showing that Cvx is a potent plateletactivator of both human and mouse platelets. Expression of recombinantGPVI at the cell surface may have been facilitated by the coexpressionof the FcRγ chain in these cells. It has been shown previously thatexpression of the FcRγ chain is required for surface expression of theFcγRI, FcγRIII, FcεRI, and for activation of platelets by collagen(Poole et al., 1997, EMBO J. 16(9):2333-2341) in mice lacking the FcRγ.

E. Cell Adhesion

Since GPVI was expressed at the cell surface of transfected cells, itscapacity to promote cell adhesion in a static system, to eitherimmobilized Cvx or collagen, was tested, and then this result wascompared this to immobilized BSA.

Materials & Methods

Cell adhesion: Collagen type I (2 μg, Chrono-log corp. Haverton, Pa.),Cvx (1.4 μg) or BSA (2 μg, Sigma, St Louis, Mo.) in 100 μl PBS wereimmobilized on Immulon II plates (Dynatech, St Cloud, France) overnightat 4° C. Plates were then saturated with 2 mg/ml BSA in PBS for one hourand washed with PBS. Cells in culture medium were labeled with ⁵¹Cr (CISBio International, Gif sur Yvette, France) for one hour at 37° C. aftercentrifugation at 150 g for 10 min, cells were washed with Hanks buffercontaining BSA (2 mg/ml) and resuspended in the same buffer. Cells wereadded to the wells. After 60 min. at room temperature, wells wereemptied and washed and the samples counted for ⁵¹Cr.

Results

Two cell lines were tested: U937 and FDC-P1. Neither the cellsexpressing GPVI, nor the control cells bound to immobilized BSA.However, expression of recombinant human or mouse GPVI in U937 orFDCP-1, respectively, clearly promotes the adhesion of these cells toimmobilized collagen and to a greater extent to immobilized Cvx (FIG.16). This result indicates that GPVI protein functions as a receptor forcollagen I. In addition, GPVI is a receptor for collagen III.

F. Association of Recombinant GPVI with FcRγ Chain

To analyze whether recombinant GPVI was expressed associated with FcRγchain we performed immunoprecipitation studies with an anti-FcRγpolyclonal antibody on lysates of U937 transduced cells compared toplatelets.

Materials & Methods

Protein analysis: The different cells (platelets, megakaryocytes andcell lines) were lysed in a buffer composed of 12 mM Tris, 300 mM NaCl,12 mM EDTA, containing 2 μM leupeptin, 2 mM PMSF, 5 KIU aprotinin, and0.2% (v/v) NP40. After 20 min at 4° C. under agitation, samples werecentrifuged at 13,000 g for 15 min at 4° C. and the supernatants wasfrozen at −80° C. Protein concentration was determined using the Bio-Radprotein assay (Bio-Rad, Ivry-sur-seine, France). For blottingexperiments, proteins were further solubilized with 2% SDS for 5 min at100° C. Proteins were separated by electrophoresis on acrylamide slabgels (Mini protean II, Bio-Rad Laboratories, Ivry-sur-seine, France) andtransferred on PVDF. Membranes were soaked with 5% non-fat dry milk andincubated with ¹²⁵I-Cvx (6×10³ Bq/ml) in PBS pH 7.4 containing 0.1%(v/v) tween 20, or with anti-GPVI IgGs (9 μg/ml in PBS pH 8, containing0.02% (v/v) tween 20 in the absence or the presence of 0.5 μM cold Cvx.Anti-GPVI IgGs were obtained as previously described (Jandrot-Perrus etal., 1997, J. Biol. Chem. 272:27035-27041) from the patient's plasmakindly provided by Pr. M. Okuma (Kyoto, Japan). Antibodies were revealedusing peroxydase-coupled protein A (Amersham Pharmacia Biotech, Uppsala,Sweden) and enhanced chemiluminescence (Amersham Pharmacia Biotech,Uppsala, Sweden). For immunoprecipitations, cell lysates were preclearedby incubation with protein A-sepharose at 4° C. for 30 min. andcentrifugation. Cleared lysates were incubated overnight at 4° C. with10 μg/ml polyclonal anti FcRγ chain antibodies (Upstate Biotechnology,NY) followed by the addition of protein A/G-sepharose (PharmaciaBiotech, Uppsala, Sweden) for 2 hours at room temperature.Immunoprecipitated proteins were eluted with 2% SDS and subjected toSDS-PAGE followed by blotting onto PVDF membranes and then probed usinganti-GPVI and anti-FcRγ chain antibodies as described above.

Results

FIG. 17 shows the analysis of the precipitated proteins byimmunoblotting with a mixture of anti- FcRγ antibodies and an IgGpreparation containing antibodies directed against GPVI. Three bandswere observed in all samples: a high molecular weight band correspondingto IgGs, a ˜50 kDa non-identified band and a 14 kDa doubletcorresponding to FcRγ chain. In addition, one band corresponding to GPVIis present in platelets and is also observed in U937 transduced with theGPVI virus but not in cell transduced with the control virus, indicatingthat recombinant GPVI is physically associated with FcRγ chain. As withFcαRI, the linkage probably involves charged residues within thetransmembrane domain: R272 or R270 respectively for hGPVI and mGPVI andD11 in the FcRγ chain.

G. Inhibition of Collagen and Cvx-induced Platelet Activation byrhusGPVI:Fc

An Fc fusion human soluble GPVI (rhusGPVI:Fc) protein was produced andpurified to investigate its ability to compete with membrane-boundplatelet GPVI.

Materials & Methods

Protein preparation: The open-reading frame of the predictedextracellular domain of T268 was PCR amplified from the Kozak sequencebefore the first methionine to asparagine 269 immediately prior to thepredicted transmembrane sequence. The PCR fragment was ligated into apCDM8 host vector containing the genomic sequence of the human IgG1 Fcdomain such that the extracellular part of the hGPVI cDNA was fused atits C-terminus via a 3 alanine linker to the hFc sequence. The sequencedDNA construct was transiently transfected into HEK 293T cells in 150 mmplates using Lipofectarmine (Gibco/BRL, Grand Island, N.Y.) according tothe manufacturer's protocol. After 72 hour post-transfection, theserum-free conditioned medium (OptiMEM, Gibco/BRL, Grand Island, N.Y.)was harvested, spun and filtered. The cells were refed with fresh mediumand harvested as above a further 72 hours later. Analysis ofsupernatants on Western blot after reducing SDS-PAGE using an anti-humanIgG Fc polyclonal antibody showed significant amounts of the recombinanthuman soluble GPVI fusion protein (rhusGPVI:Fc) in the supernatants witha relative molecular mass of approximately 75-80 kDa relative to Mark 12molecular weight standards cocktail (Novex, San Diego, Calif.).

The conditioned media was passed over a Prosep-G protein G column (10mL, Bioprocessing Inc., Princeton, N.J.); the column was then washedwith PBS, pH 7.4 and eluted with 200 mM glycine, pH 3.0 at 7 mL/min.Fractions from the 280 nm elution peak containing human rhusGPVI:Fc werepooled and dialyzed in 8000 MWCO dialysis tubing against 2 changes of 4LPBS, pH 7.4 at 4° C. with constant stirring. The buffered exchangedmaterial was then sterile filtered (0.2 _m, Millipore Corporation,Bedford. Mass.) and frozen at −80° C.

Platelet preparation: Blood from healthy volunteers was collected byvenepuncture on acid-citrate-dextrose anticoagulant (ACD-A). Whenneeded, platelets were labeled by incubating the platelet rich plasma(PRP) with 0.6 μM (¹⁴C) 5-hydroxytryptamine for 30 min. at 37° C.Platelet pellets were obtained by centrifugation of the platelet richplasma (PRP) and were washed two times as previously described(Jandrot-Perrus et al., 1997, J. Biol. Chem. 272:27035-27041).

Platelet aggregation and secretion: Aggregation of washed platelets(3×10⁸/ml) in reaction buffer was initiated by collagen type I (Bio/Datacorp, Horsham, Pa.) or Cvx. Experiments were performed with stirring at37° C. in a Chrono-Log aggregometer (Chrono-log corp. Haverton, Pa.).Release of (¹⁴C) 5-hydroxytryptamine was measured as describedpreviously (Jandrot-Perrus et al., 1997, J. Biol. Chem.272:27035-27041).

Results

rhusGPVI:Fc did not induce platelet aggregation or granule secretion byitself. When platelets were incubated with Cvx, addition of rhusGPVI:Fc(0.25 to 5 μg/ml) fully inhibited platelet aggregation and dense granulesecretion (FIG. 18). In addition, when rhusGPVI:Fc was added to theplatelet suspension prior to Cvx, it also inhibited aggregation andsecretion, indicating that it could compete with platelet GPVI for Cvx(FIG. 18A). Incubation of collagen with rhusGPVI:Fc induces a loss inits ability to induce platelet aggregation and secretion (FIG. 18B).However, a tenfold higher concentration of rhusGPVI:Fc than required forCvx was needed to produce this inhibitory effect. Furthermore, whenrecombinant soluble GPVI was added to platelets prior to collagen, noinhibition was observed (FIG. 18B). These results demonstrate that theextracellular domain of GPVI is active in blocking Cvx- andcollagen-induced platelet aggregation.

Discussion

GPVI, despite its essential role in collagen-induced plateletaggregation, is described as having a minor role in platelet adhesion tocollagen. Other receptors such as the GPIb-IX-V complex or the integrinα2β1 are major players responsible for platelet adhesion to collagen.However, immobilized Cvx is able to induce platelet adhesion indicatingthat GPVI may be involved in adhesion in these conditions. The aboveresults demonstrate that expression of GPVI in U937 and FDCP-1 cellsinduces cell adhesion to a collagen- or Cvx-coated surface. The numberof cells that bound to immobilized Cvx was significantly higher thanthose bound to collagen. This result indicates differences in thedensity of GPVI binding sites on the two surfaces. Cvx is a pure GPVIligand and when immobilized it produces a highly reactive surface whileGPVI binding sites should be disseminated on collagen fibers resultingin a less reactive surface.

Nevertheless, these results indicate that recombinant GPVI mimics thephysiological function of platelet GPVI (i.e., binding to collagen). Thedifference in reactivity between collagen and Cvx is further emphasizedby the differences in the inhibitory effect that the recombinant solubleGPVI has on collagen and Cvx-induced platelet activation. Indeed,soluble recombinant GPVI inhibits Cvx-induced platelet activation in theabsence of preincubation with Cvx whilst it requires a preincubationwith collagen to inhibit collagen-induced platelet activation. Thisprobably reflects the rapid kinetics of interaction between GPVI and Cvxcompared to those between GPVI and collagen. The affinity of recombinantsoluble GPVI for Cvx is probably very high for two reasons: (i) solubleGPVI is expressed in a divalent Fc fusion form and (ii) Cvx ismultivalent due to its hexameric structure. Thus, GPVI binding sites oncollagen fibers are probably dispersed and poorly accessible.Alternatively, these observations could also suggest that binding ofcollagen to its other receptors, including the integrin α2β1, promotesits subsequent interaction with GPVI.

GPVI plays an important role in the development of thrombi probablybecause it is the receptor that appears to govern platelet activation atthe contact of collagen and thus which induces platelet recruitment.Indeed, patients with GPVI deficiency or anti-GPVI-containing seradisplayed bleeding disorders (see Background, above). The molecularcloning of GPVI provides the opportunity to characterize the mechanismof these deficiencies, the precise interaction between GPVI and theintegrin α2β1 in collagen-induced platelet activation but also the roleof GPVI in thromboembolic diseases. GPIIb-IIIa (integrin αIIb P3) is theonly platelet receptor against which efficient antagonists have been sofar developed (Lefkovits et al., 1995, N. Engl. J. Med. 332:1553-9).Even if GPIIb-IIIa may be involved in platelet adhesion, its principalrole is to bind fibrinogen allowing platelet aggregation and serving asthe final common pathway of platelet thrombus formation regardless ofthe metabolic pathway initiating platelet activation. In contrast, GPVIis involved in an early step of platelet activation occurringimmediately when platelets enter in contact with the subendothelialmatrix.

GPVI represents an alternative and more specific target for newanti-thrombotic compounds. Antagonist can be directed against either ofthe two players, i.e., collagen GPVI binding sites or GPVI itself.Because these observations suggest that the GPVI binding sites are noteasily accessible on collagen fibers, an antagonist directed againstGPVI may be more efficient than an antagonist directed against collagen.

H. Bleeding Time of Mice Transplanted with Bone Marrow Cells ExpressingGPVI

The results presented herein support the role of GPVI in the formationof platelet aggregates and the development of a hemostatic plug.

Materials & Methods

Isolation of Lin⁻ bone marrow cells: Bone marrow cells were collectedfrom mice that had been administered 150 mg/kg of 5-fluorouracil (5-FU)intravenously for four days. The cells were resuspended in phosphatebuffered saline (PBS), 0.5% fetal calf serum (FCS) and incubated for 20minutes at 4° C. with a mixture of four fluorescent fluoresceinisothiocyanate (FITC)-conjugated rat monoclonal antibodies directedagainst mouse CD3e, CD11b, CD45R, and Ly-6G (Pharmingen, San Diego,Calif.). After labeling, cells were washed and incubated at 4° C. withanti-FITC microbeads (Miltenyi Biotech, Auburn, Calif.). After a 15minute incubation, cells were washed, filtered through a large pore sizefilter and applied onto a magnetic cell sorting depletion column (typesBS, Miltenyi Biotech) held onto a magnetic separator (Super MACS,Miltenyi Biotech). Depletion of the magnetically labeled cells (lineagepositive) out of the bone marrow sample was done according to themanufacturer's instructions. In some experiments, Sca-1⁺/Lin⁻ cells wereisolated using a Sca-1 multiSort Kit (Miltenyi Biotech). Afterseparation, cells (Lin⁻ or Sca-1⁺/Lin⁻) were washed and resuspended inDMEM, 10% FCS (Stem Cell Technologies, Vancouver, Canada).

Infection procedure: Isolated Lin⁻ or Sca-1⁺/Lin⁻ bone marrow cells werestimulated at 37° C., 10% CO² with 10 ng/ml of recombinant mouseinterleukin-3 (rmIL-3; Endogen, Woburn, Mass.), 10 ng/ml recombinantmouse interleukin-6 (rmIL-6; Endogen), 100 ng/ml recombinant mouse stemcell factor (rmSCF; R&D Systems, Inc., Mineapolis, Minn.), 100 ng/mlrecombinant mouse fms-like tyrosine kinase-3 ligand (rmFlt-3L; R&DSystems, Inc), and 1% of a conditioned medium containing mousethrombopoietin (mTPO). The mTPO conditioned medium (containingapproximately 10⁴ U/ml of mTPO) was collected from confluentMPZenTPO-virus producing cells, filtered and virus-inactivated at 56° C.for 1 hour. After two days of stimulation, bone marrow cells wereinfected with recombinant retrovirus containing the cDNA encoding murinefull length GPVI, recombinant retrovirus containing the cDNA encodingthe extracellular domain of murine GPVI, or a control retrovirus.

Bleeding assay: Infected bone marrow cells were transplanted intolethally irradiated mice, and two months post-transplantation mice wereanalyzed for the recovery time from a small tail vein incision. Theblood flow from the incision was measured at 37° C. in saline.

Results and Discussion:

FIG. 19 depicts the bleeding time of lethally irradiated micetransplanted with bone marrow cells engineered to express full lengthGPVI, the extracellular domain of GPVI, or a control. Mice transplantedwith bone marrow cells engineered to express the extracellular domain ofGPVI, a soluble product, had the longest recovery time from a small tailvein incision. No significant difference in the recovery time from anincision in the tail vein was observed in mice transplanted with bonemarrow cells engineered to express the full length GPVI from thecontrol. These results suggest that the soluble form of GPVI inhibitsthe collagen-platelet interaction necessary for platelet aggregation anddevelopment of a hemostatic plug.

Uses of TANGO 268 Nucleic Acids, Polypeptides, and Modulators Thereof

As TANGO 268 was originally found in an megakaryocyte library, and inlight of the fact that TANGO 268 has been shown herein to be GPVI, TANGO268 nucleic acids, proteins, and modulators thereof can be used tomodulate the proliferation, morphology, migration, differentiation,and/or function of megakaryocytes and platelets, including duringdevelopment, e.g., embryogenesis. TANGO 268 nucleic acids, proteins, andmodulators thereof can also be used to modulate leukocyte-platelet andplatelet-endothelium interactions in inflammation and/or thrombosis.Further, TANGO 268 nucleic acids, proteins, and modulators thereof canbe used to modulate platelet aggregation and degranulation. For example,antagonists to TANGO 268 action, such as peptides, antibodies or smallmolecules that decrease or block TANGO 268 binding to extracellularmatrix components (e.g., collagen or integrins) or antibodies preventingTANGO 268 signaling, can be used as collagen or platelet release andaggregation blockers. In a specific example, a polypeptide comprisingthe extracellular domain of TANGO 268 can be used to decrease or blockTANGO 268 binding to extracellular matrix components (i.e., collagen),or to prevent platelet aggregation. In another example, agonists thatmimic TANGO 268 activity, such as peptides, antibodies or smallmolecules, can be used to induce platelet release and aggregation.

In further light of the fact that TANGO 268 represents GPVI, and itsexpression is restricted to cells of the megakaryocyte lineage, TANGO268 nucleic acids, proteins, and modulators thereof can be used tomodulate disorders associated with abnormal or aberrant megakaryocyteand/or platelet proliferation, differentiation, morphology, migration,aggregation, degranulation and/or function. Examples of these disordersinclude, but are not limited to, bleeding disorders (e.g., bleedingtendency and/or prolonged bleeding time) such as thrombocytopenia (e.g.,idiopathic thrombocytopenic purpura (ITP) or immune thrombocytopenia orthrombocytopenia induced by chemotherapy or radiation therapy).

As TANGO 268 represents GPVI, and GPVI is a component in processesinvolving platelet binding to the vascular subendothelium, plateletactivation and inflammation processes, TANGO 268 nucleic acids,proteins, and modulators thereof can be used to modulate thromboticdisorders (e.g., thrombotic occlusion of coronary arteries), hemorrhagicdisorders, diseases exhibiting quantitative or qualitative plateletdysfunction and diseases displaying endothelial dysfunction(endotheliopathies). These diseases include, but are not limited to,coronary artery and cerebral artery diseases. Further, TANGO 268 nucleicacids, proteins, and modulators thereof can be used to modulate cerebralvascular diseases, including stroke and ischemia, venous thromboembolismdiseases (e.g., diseases involving leg swelling, pain and ulceration,pulmonary embolism, abdominal venous thrombosis), thromboticmicroangiopathies, vascular purpura, and GPVI deficiencies as described,e.g., in Moroi and Jung, 1997, Thrombosis and Haemostasis 78:439-444.TANGO 268 nucleic acids, proteins, and modulators thereof can be used tomodulate symptoms associated with platelet disorders and/or diseases(e.g., bleeding disorders). In particular, TANGO 268 nucleic acids,proteins, and modulators thereof can be used to modulate symptomsassociated with ITP such as purpura and severe bleeding problems.

As GPVI has been shown to be important for platelet adhesion andaggregation, and platelet adhesion and aggregation play an importantrole in acute coronary diseases, TANGO 268 nucleic acids, proteins andmodulators thereof can be used to modulate coronary diseases (e.g.,cardiovascular diseases including unstable angina pectoris, myocardialinfarction, acute myocardial infarction, coronary artery disease,coronary revascularization, coronary restenosis, ventricularthromboembolism, atherosclerosis, coronary artery disease (e.g.,arterial occlusive disorders), plaque formation, cardiac ischemia,including complications related to coronary procedures, such aspercutaneous coronary artery angioplasty (balloon angioplasty)procedures). With respect to coronary procedures, such modulation can beachieved via administration of GPVI modulators prior to, during, orsubsequent to the procedure. In a preferred embodiment, suchadministration can be utilized to prevent acture cardiac ischemiafollowing angioplasty.

TANGO 268 nucleic acids, proteins and modulators thereof can, therefore,be used to modulate disorders resulting from any blood vessel insultthat can result in platelet aggregation. Such blood vessel insultsinclude, but are not limited to, vessel wall injury, such as vesselinjuries that result in a highly thrombogenic surface exposed within anotherwise intact blood vessel e.g., vessel wall injuries that result inrelease of ADP, thrombin and/or epinephrine, fluid shear stress thatoccurs at the site of vessel narrowing, ruptures and/or tears at thesites of atherosclerotic plaques, and injury resulting from balloonangioplasty or atherectomy.

Preferably, the TANGO 268 nucleic acids, proteins and modulators (e.g.,anti-TANGO 268 antibodies) thereof do not effect initial plateletadhesion to vessel surfaces, or effect such adhesion to a relativelylesser extent than the effect on platelet-platelet aggregation, e.g.,unregulated platelet-platelet aggregation, following the initialplatelet adhesion. Further, in certain embodiments, it is preferred thatthe TANGO 268 nucleic acids, proteins and modulators (e.g., anti-TANGO268 antibodies) thereof do not effect other platelet attributes orfunctions, such as agonist-induced platelet shape change (e.g.,GPIb-vWF-mediated platelet agglutination induced by ristocetin), releaseof internal platelet granule components, activation of signaltransduction pathways or induction of calcium mobilization upon plateletactivation.

Further, polymorphisms associated with particular TANGO 268 alleles,such as those in platelet receptor glycoprotein Ia/IIa that areassociated with risk of coronary disease (see, e.g., Moshfegh et al.,1999, Lancet 353:351-354), can be used as a marker to diagnose abnormalcoronary function (e.g., coronary diseases such as myocardialinfarction, atherosclerosis, coronary artery disease, plaque formation).

In further light of the fact that TANGO 268 is GPVI, TANGO 268 nucleicacids, proteins and modulators thereof can be used to modulate disordersassociated with aberrant signal transduction in response to collagen orother extracellular matrix proteins.

In addition to the above, TANGO 268 nucleic acids, proteins andmodulators thereof can be utilized to modulate disorders associated withaberrant levels of TANGO 268 expression and/or activity either in cellsthat normally express TANGO 268 or in cells that do not express TANGO268. For example, TANGO 268 nucleic acids, proteins and modulatorsthereof can be used to modulate disorders associated with aberrantexpression of TANGO 268 in cancerous (e.g., tumor) cells that do notnormally express TANGO 268. Such disorders can include, for example,ones associated with tumor cell migration and progression to metastasis.

In light of the fact that TANGO 268 (i.e., GPVI) has been shown tointeract with collagen, and the progression, migration and metastasis ofcancer cells has been shown to correlate with the attachment of cancercells to interstitial collagen (see, e.g., Martin et al., 1996, Int. J.Cancer 65:796-804), abnormal and/or aberrant TANGO 268 expression (e.g.,expression of TANGO 268 in cells, such as tumor cells, that do notnormally express it or increased expression of TANGO 268 in cells thatdo normally express it) can be used as a marker for the progression,migration and metastasis of cancerous cells. In particular, abnormaland/or aberrant TANGO 268 expression can be used as a marker for theprogression, migration and metastasis of colon cancer and liver cancer.

In light of TANGO 268 exhibiting homology to human monocyte inhibitoryreceptor, TANGO 268 nucleic acids, proteins and modulators thereof canbe used mediate the downregulation of cell activation via phosphatases.In light of TANGO 268 containing two Ig-like domains, TANGO 268 nucleicacids, proteins and modulators thereof can be used to modulateimmunoregulatory functions. Further, as TANGO 268 is expressed in theliver, embryo, bone marrow, and peripheral blood, TANGO 268 nucleicacids, proteins, and modulators thereof can be used to treat disordersof these cells, tissues or organs, e.g., liver disorders andimmunological disorders.

TANGO 268 is expressed on the surface of platelets. As such, a cellularand therapeutic target of modulators of TANGO 268 (e.g., anti-TANGO 268antibodies) is readily available for testing and analysis (e.g., for invitro testing and analysis). This coupled with the availability ofseveral different relevant platelet assays (see below) provides anunusual drug development opportunity for TANGO 268 modulators. Forexample, the in vivo pharmacodynamic characterization of TANGO 268modulators can be facilitated via the availability of various plateletassays (e.g., prolongation of bleeding time, quantitative measurement ofTANGO 268 receptor blockade, inhibition of ex vivo platelet aggregation)that can be correlated with each other to permit more effectiveassessment of a modulator's functional consequences. The correlationavailable for such assays, therefore, allows for the in vitrocharacterization of a TANGO 268 modulator to more directly apply to themeasurement of the modulator's therapeutic effect.

In addition to utilizing the availability of platelets and plateletassays for assessing the therapeutic efficacy, including clinicalefficacy, of a TANGO 268 modulator, this availability can also beutilized for preclinical drug development aspects such as determiningmodulator dosage response, toxicology, magnitude of effect (e.g.,magnitude of initial effect and magnitude of effect's duration),function, specificity (e.g., specificity with respect to particularplatelet functions), receptor specificity, and species specificity(which, in turn, can identify appropriate animal models for pharmacologystudies).

Modulators of TANGO 268 platelet aggregation can also be utilized, e.g.,for ex vivo procedures, e.g., ex vivo inhibition of plateletaggregation.

Assays for the Detection of TANGO 268 Expression or Activity

The expression of TANGO 268 can be readily detected, e.g., byquantifying TANGO 268 protein and/or RNA. Many methods standard in theart can be thus employed, including, but not limited to, immunoassays todetect and/or visualize gene expression (e.g., Western blot,immunoprecipitation followed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE), immunocytochemistry, etc) and/orhybridization assays to detect gene expression by detecting and/orvisualizing respectively mRNA encoding a gene (e.g., Northern assays,dot blots, in situ hybridization, etc), etc. Ligand binding assays, suchas described above, can be performed to assess the function of TANGO268.

The activity of a TANGO 268 protein can be measured by employing methodsknown to those of skill in the art. For example, the activity of a TANGO268 protein can be analyzed by treating of platelets or TANGO268-transfected cells with collagen or convulxin and measuring theeffect of such treatment on the level of tyrosine phosphorylation ofsignaling molecules, such as FcRγ, Syk, and PLCγ2 (e.g., tyrosinephosphorylation can be detected by immunoprecipitation followed bySDS-PAGE, kinase assays, etc.). The activity of a TANGO 268 protein canalso be analyzed by measuring changes in the concentration of freeintracellular Ca²⁺ induced by the treatment of platelets or TANGO 268transfected cells with collagen or convulxin. Briefly, platelets orTANGO 268 transfected cells are incubated with fura-2 fluorescence at37° C. and, then incubated with 2 mM CaCl₂ prior to incubation withconvulxin, collagen or thrombin (an agent that does not activate TANGO268). The cells are lysed in lysis buffer, and the concentration of freeintracellular Ca²⁺ is measured by fluorescence at 37° C. using aspectrophotometer (see, e.g., Jandrot-Perrus et al., 1997, J. of Biol.Chem. 272:27035-27041).

The activity of a TANGO 268 protein can also be analyzed by a plateletadhesion assay. Briefly, the adhesion assay is performed as follows:⁵¹Cr-labeled platelets are incubated in microtiter plates that havecollagen, convulxin or BSA immobilized to the surface of the wells, thecells are washed, 2% SDS is added to each well, and the number ofadherent platelets is determined by counts for ⁵¹Cr using ascintillation counter (see, e.g., Jandrot-Perrus et al., 1997, J. ofBiol. Chem. 272:27035-27041). Further, the activity of a TANGO 268protein can be analyzed by platelet aggregation assays or secretionassays known to those of skill in the art (see, e.g., Moroi et al.,1989, J. Clin. Invest. 84:1440-1445 and Poole et al., 1997, EMBO J.16(9):2333-2341). Briefly, the platelet aggregation is performed asfollows: platelets are incubated with collagen or convulxin in a cuvetteat 37° C. while being stirred, and the cell suspension is monitored by alumiaggregometer.

Such assays may be utilized as part of TANGO 268 diagnostic assays. Inaddition, such assays may be utilized as part of screening methods foridentifying compounds that modulate the activity and/or expression ofTANGO 268.

Assays for the Function of TANGO 268

The function of a TANGO 268 protein can be analyzed by transplantinghematopoietic cells engineered to express TANGO 268 or a control intolethally irradiated mice. The affect of TANGO 268 expression on thefunction, development and proliferation of hematopoietic cells,specifically platelets, can be determined by comparing mice transplantedwith hematopoietic cells expressing TANGO 268 to mice transplanted withhematopoietic cells expressing a control. For example, the role of TANGO268 in platelet aggregation can be analyzed by transplanting mice withhematopoietic cells engineered to express TANGO 268 or fragmentsthereof. The irradiated mice may be normal, transgenic or knockout mice,and the hematopoietic cells may be obtained from normal, transgenic orknockout mice.

The efficacy of using TANGO 268 nucleic acids, proteins or modulatorsthereof to modulate the expression of a given gene can be analyzed usingirradiated mice transplanted with hematopoietic cells engineered toexpress TANGO 268 or modulators thereof. The affect of TANGO 268 nucleicacids, proteins or modulators thereof on the expression of a gene ofinterest can be measured by analyzing the RNA or protein levels of thegene of interest. Techniques known to those of skill can be used tomeasure RNA and protein levels in vivo and in vitro. For example, RNAexpression can be detected in vivo by in situ hybridization. Further,the efficacy of using TANGO 268 nucleic acids, proteins or modulatorsthereof to treat, inhibit or prevent a particular disease or disordercan be analyzed by using irradiated mouse or rat models of a disease ordisorder transplanted with hematopoietic cells engineered to expressTANGO 268 or modulators thereof.

Assays for Analysis of TANGO 268 Modulators

A variety of assays can be utilized to analyze a TANGO 268 protein,nucleic acid or modulator thereof. Such assays can include in vivo, exvivo and in vitro assays, as described herein. See, also, e.g., Loscalzoand Schaefer (eds), 1998, Thrombosis and Hemorrhage 2^(nd) Edition,Chapter 16, Williams and Wilkins: Baltimore, Md.; Horton (ed), 1995,Adhesion Receptors as Therapeutic Targets, Chapter 15, CRC Press, Inc.:London, United Kingdom, and U.S. Pat. No. 5,976,532.

For example, in view of the fact that TANGO 268 is a cell surfacereceptor, in particular, a platelet receptor, standard quantitativebinding studies can be utilized to measure modulator binding toplatelets. Horton (ed), 1995, Adhesion Receptors as Therapeutic Targets,Chapter 15, CRC Press, Inc.: London, United Kingdom. Such binding assayscan also be utilized to perform receptor blockade studies to measure thenumber of cellular sites available for binding modulator by comparingthe number of molecules of labeled modulator molecules (e.g., labeledanti-TANGO 268 antibodies) bound per platelet at a series ofconcentrations with the number of modulator molecules bound atsaturation. See, e.g., Coller et al., 1985, J. Clin. Invest. 76: 101 orU.S. Pat. No. 5,854,005.

The reversibility of modulator molecule (e.g., anti-TANGO 268antibodies) binding on platelets can also be tested, using, e.g.,techniques such as those described in Coller et al., 1985, J. Clin.Invest. 76: 101, and U.S. Pat. No. 5,976,532. In addition, undernon-competitive conditions, the rate of modulator dissociation can beassessed by, e.g., flow cytometry analysis of platelets whenfluorescently labeled modulator (e.g., anti-TANGO 268 antibody)-coatedplatelets are mixed with an equal number of untreated platelets andincubated at physiological temperature. In instances wherein appreciablereversibility indicates that inhibitory effects of single in vivoinjection can be relatively short-lived, suggesting that anadministration regimien involving an initial bolus followed bycontinuous infusion may be most effective.

In vitro and ex vivo assays for inhibition of platelet aggregation canalso be utilized. Such assays are well known to those of skill in theart and include, but are not limited to the turbidometric method, inwhich aggregation is measured as an increase in transmission of visiblelight through a stirred or agitated platelet suspension. See, e.g.,Chanarin, L., 1989, Laboratory Haematology, Chapter 30, Churchill,Livingstone, London; and Schmidt, R. M. (ed), 1979, CRC Handbook Seriesin Clinical Laboratory Science, CRC Press, Inc.: Boca Raton, Fla.

Platelet aggregation can also be assayed via methods such as thosedescribed in U.S. Pat. No. 5,976,532. For example, in a non-limitingexample of such a method, the platelet concentration in platelet-richplasma obtained (PRP) obtained from normal or patient blood samples isadjusted to 200,000 to 300,000/mm³. In an in vitro assay, the PRP isaliquoted and incubated in the presence or absence of a TANGO 268modulator (e.g., an anti-GPVI antibody) for a period of time (e.g., 15minutes at 37° C.) prior to the addition of a platelet inducing agonist(e.g., ADP, thrombin, collagen, epinephrine, and ristocetin). In an exvivo assay, the PRP obtained from individuals treated with TANGO 268 ora placebo is aliquoted and incubated in the presence of a plateletinducing agonist (e.g., ADP, thrombin, collagen, epinephrine, andristocetin). Platelet aggregation is measured by assessing an increasein the transmission of visible light through a platelet suspension usinga spectrophotometer.

In certain embodiments, it is preferred that the TANGO 268 modulator noteffect platelet attributes or functions other than platelet aggregation.Such other platelet attributes or functions, include, for example,agonist-induced platelet shape change (e.g., GPIb-vWF-mediated plateletagglutination induced by ristocetin), release of internal plateletgranule components, activation of signal transduction pathways orinduction of calcium mobilization upon platelet activation. Assays forthese platelet attributes and functions are well known to those of skillin the art and can be utilized to routinely test, develop and identifyTANGO 268 modulators exhibiting a specificity for modulation of plateletaggregation.

The shape of a platelet can be analyzed in any in vitro assay known tothose of skill in the art. Briefly, platelets are contacted in thepresence or absence of a TANGO 268 modulator with a platelet inducingagonist (e.g., ADP, thrombin, collagen, epinephrine, and ristocetin) andthe shape of the platelets are assessed by microscopy or by flowcytometry. Platelet degranulation can be analyzed, for example, bymeasuring the presence of ATP in vitro following stimulation with aplatelet inducing agonist in the presence or absence of a TANGO 268modulator (see, e.g., Loscalzo and Schaefer (eds), 1998, Thrombosis andHemorrhage 2^(nd) Edition, Chapter 16, Williams and Wilkins: Baltimore,Maryland). The activation of platelet signal transduction pathways canbe analyzed in in vitro and ex vivo assays using assays known to thoseof skill in the art. For example, the activation of signal transductionpathways in vitro can be analyzed by contacting platelet-rich plasmasamples with platelet agonists (e.g., collagen and convulxin) in thepresence or absence of a TANGO 268 modulator and measuring the effect ofsuch treatment on the level of tyrosine phosphorylation of signalingmolecules, such as FcRγ, Syk, and PLCγ 2 (e.g., tyrosine phosphorylationcan be detected by immunoprecipitation followed by SDS-PAGE, kinaseassays, etc.). In an ex vivo assay, the activation of signaltransduction pathways can be analyzed by contacting platelet-rich plasmasamples obtained from individuals treated with TANGO 268 or a placebowith a platelet agonist (e.g., collagen and convulxin) and measuring theeffect of such treatment on the level of tyrosine phosphorylation ofsignaling molecules, such as FcRγ, Syk, and PLCγ 2 (e.g., tyrosinephosphorylation can be detected by immunoprecipitation followed bySDS-PAGE, kinase assays, etc.). The effect of platelet activation oncalcium mobilization can also be analyzed by measuring changes in theconcentration of free intracellular Ca²⁺ induced in in vitro and ex vivoassays using assays known to those of skill in the art. Briefly,platelet-rich platelets are incubated with fura-2 fluorescence at 37° C.and then incubated with 2 mM CaCl₂ in the presence or absence of a TANGO268 modulator prior to incubation with a platelet agonist (e.g.,convulxin, collagen and thrombin). The cells are lysed in lysis buffer,and the concentration of free intracellular Ca²⁺ is measured byfluorescence at 37° C. using a spectrophotometer (see, e.g.,Jandrot-Perrus et al., 1997, J. of Biol. Chem. 272:27035-27041).

Other assays for platelets include, in vivo assays such as assessment ofprolongation of bleeding time. For example, the bleeding time resultingfrom an injury (e.g., a small tail vein incision) in an animal modeltreated with a TANGO 268 modulator can be compared to an animal modeltreated with a placebo. In humans, the number of bleeding episodes andthe length of the bleeding time during a bleeding episode for a humantreated with a TANGO 268 modulator can be compared to a human treatedwith a placebo.

The efficacy of TANGO 268 modulators can be assessed in a variety ofanimal models of arterial thrombosis, including, but not limited to, theFolts model, the electrolytic injury model, the thrombin-inducedarterial thrombosis model, and a model of acute thrombosis resultingfrom injury induced by coronary balloon angioplasty (see, e.g., Loscalzoand Schaefer (eds), 1998, Thrombosis and Hemorrhage 2^(nd) Edition,Chapter 16, Williams and Wilkins: Baltimore, Md.). The Folts model,which is the most widely used animal model of coronary and carotidartery thrombosis, is produced by mechanical concentric vessel narrowingusing a cylinder placed around the artery. The electrolytic model, whichis used for deep arterial injury, is produced by introducing an electriccurrent via an electrode to the intimal layer of a stenosed vessel. Byapplying species specificity data that can readily be obtained using,e.g., the platelet aggregation assays described herein, animal modelsparticularly well suited to study of any given TANGO 268 modulator canbe chosen.

Tables 1 and 2 below provide a summary of the sequence information forTANGO 268. TABLE 1 Summary of TANGO 268 Sequence Information AccessionGene cDNA ORF Figure Number Human SEQ ID NO: 1 SEQ ID NO: 2 207180 TANGO268 Mouse SEQ ID NO: 14 SEQ ID NO: 15 PTA-225 TANGO 268

TABLE 2 Summary of Domains of TANGO 268 Proteins Signal Mature ProteinSequence Protein Extracellular Ig-like Transmembrane Cytoplasmic HUMANaa 1-20 of aa 21-339 of aa 21-269 of aa 48-88; aa 270-288 of aa 289-339of TANGO SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 3 134-180 of SEQ ID NO: 3SEQ ID NO: 3 268 (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 9) SEQ ID NO:3 (SEQ ID NO: 8) (SEQ ID NO: 10) (SEQ ID NO: 6; SEQ ID NO: 7) MOUSE aa1-21 of aa 22-313 of aa 22-267 of aa 49-89; aa 268-286 of aa 287-313 ofTANGO SEQ ID NO: 16 SEQ ID NO: 16 SEQ ID NO: 16 135-181 of SEQ ID NO: 16SEQ ID NO: 16 268 (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) SEQ IDNO: 16 (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22; SEQ ID NO: 23)Various aspects of the invention are described in further detail in thefollowing subsections:I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a polypeptide of the invention or a biologically activeportion thereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encoding apolypeptide of the invention and fragments of such nucleic acidmolecules suitable for use as PCR primers for the amplification ormutation of nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. As used herein, the term“isolated”when referring to a nucleic acid molecule does not include anisolated chromosome.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 2, 14, or 15 ora complement thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all or aportion of the nucleic acid sequences of SEQ ID NO:1, 2, 14 or 15 as ahybridization probe, nucleic acid molecules of the invention can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., eds., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence of SEQ ID NO:1, 2, 14 or 15 or a portionthereof. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding a full length polypeptide ofthe invention for example, a fragment which can be used as a probe orprimer or a fragment encoding a biologically active portion of apolypeptide of the invention. The nucleotide sequence determined fromthe cloning one gene allows for the generation of probes and primersdesigned for use in identifying and/or cloning homologues in other celltypes, e.g., from other tissues, as well as homologues from othermammals. The probe/primer typically comprises substantially purifiedoligonucleotide. In one embodiment, the oligonucleotide comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12, preferably about 25, more preferably about 50, 75,100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides ofthe sense or anti-sense sequence of SEQ ID NO:1, 2, 14 or 15 or of anaturally occurring mutant of SEQ ID NO:1, 2, 14 or 15. In anotherembodiment, the oligonucleotide comprises a region of nucleotidesequence that hybridizes under stringent conditions to at least 400,preferably 450, 500, 530, 550, 600, 700,800, 900, 1000 or 1150consecutive oligonucleotides of the sense or antisense sequence of SEQID NO:1, 2, 14 or 15 of a naturally occurring mutant of SEQ ID:1,2 14 or15.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences encoding the sameprotein molecule encoded by a selected nucleic acid molecule. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit for identifying cells ortissues which mis-express the protein, such as by measuring levels of anucleic acid molecule encoding the protein in a sample of cells from asubject, e.g., detecting mRNA levels or determining whether a geneencoding the protein has been mutated or deleted.

A nucleic acid fragment encoding a biologically active portion of apolypeptide of the invention can be prepared by isolating a portion ofany of SEQ ID NO:3 or 16 expressing the encoded portion of thepolypeptide protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the polypeptide.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NO:1, 2, 14 or 15 due todegeneracy of the genetic code and thus encode the same protein as thatencoded by the nucleotide sequence of SEQ ID NO:3 or 16.

In addition to the nucleotide sequences of SEQ ID NO:3 or 16, it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequence may exist within apopulation (e.g., the human population). Such genetic polymorphisms mayexist among individuals within a population due to natural allelicvariation. An allele is one of a group of genes which occuralternatively at a given genetic locus. For example, human TANGO 268 hasbeen mapped to chromosome 19, and therefore TANGO 268 family members caninclude nucleotide sequence polymorphisms (e.g., nucleotide sequencesthat vary from SEQ ID NO:1 and SEQ ID NO:2) that map to this chromosomallocus (e.g., region of chromosome 19q13) and such sequences representTANGO 268 allelic variants. As used herein, the phrase “allelic variant”refers to a nucleotide sequence which occurs at a given locus or to apolypeptide encoded by the nucleotide sequence. As used herein, theterms “gene” and “recombinant gene” refer to nucleic acid moleculescomprising an open reading frame encoding a polypeptide of theinvention. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of a given gene. Alternative allelescan be identified by sequencing the gene of interest in a number ofdifferent individuals. This can be readily carried out by usinghybridization probes to identify the same genetic locus in a variety ofindividuals. Any and all such nucleotide variations and resulting aminoacid polymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention. In one embodiment, polymorphismsthat are associated with a particular disease and/or disorder are usedas markers to diagnose said disease or disorder. In a preferredembodiment, polymorphisms are used as a marker to diagnose abnormalcoronary function (e.g., coronary diseases such as myocardialinfarction, atherosclerosis, coronary artery disease, plaque formation).

Moreover, nucleic acid molecules encoding proteins of the invention fromother species (homologues), which have a nucleotide sequence whichdiffers from that of the human or mouse protein described herein areintended to be within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and homologues of a cDNA ofthe invention can be isolated based on their identity to the humannucleic acid molecule disclosed herein using the human cDNAs, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions. Forexample, a cDNA encoding a soluble form of a membrane-bound protein ofthe invention isolated based on its hybridization to a nucleic acidmolecule encoding all or part of the membrane-bound form. Likewise, acDNA encoding a membrane-bound form can be isolated based on itshybridization to a nucleic acid molecule encoding all or part of thesoluble form.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence, ofSEQ ID NO:1, or a complement thereof.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 50, 100, 200, 300, 400, 500, 600, 700, 800 or900 nucleotides in length and hybridizes under stringent conditions tothe nucleic acid molecule comprising the nucleotide sequence, preferablythe coding sequence, of SEQ ID NO:2, or a complement thereof.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 50, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400 or 1500 nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence, ofSEQ ID NO:14, or a complement thereof.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 50, 100, 200, 300, 400, 500, 600, 700, 800 or900 nucleotides in length and hybridizes under stringent conditions tothe nucleic acid molecule comprising the nucleotide sequence, preferablythe coding sequence, of SEQ ID NO:15, or a complement thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1, 2, 14 or 15, or a complement thereof,corresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention sequence that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of theprotein. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence without altering the biological activity, whereasan “essential” amino acid residue is required for biological activity.For example, amino acid residues that are not conserved or onlysemi-conserved among homologues of various species may be non-essentialfor activity and thus would be likely targets for alteration.Alternatively, amino acid residues that are conserved among thehomologues of various species (e.g., mouse and human) may be essentialfor activity and thus would not be likely targets for alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from SEQ ID NO:3, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:3.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from SEQ ID NO:16, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:16.

An isolated nucleic acid molecule encoding a variant protein can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of SEQ ID NO:1, 2, 14 or 15such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant polypeptide that is a variant of apolypeptide of the invention can be assayed for: (1) the ability to formprotein: protein interactions with proteins in a signaling pathway ofthe polypeptide of the invention; (2) the ability to bind a ligand ofthe polypeptide of the invention; or (3) the ability to bind to anintracellular target protein of the polypeptide of the invention. In yetanother preferred embodiment, the mutant polypeptide can be assayed forthe ability to modulate cellular proliferation, cellular migration orchemotaxis, or cellular differentiation.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga polypeptide of the invention, e.g., complementary to the coding strandof a double-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can be antisense to all or part of a non-coding region ofthe coding strand of a nucleotide sequence encoding a polypeptide of theinvention. The non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences which flank the coding region and are nottranslated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a selectedpolypeptide of the invention to thereby inhibit expression, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol Impromoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptide of theinvention can be designed based upon the nucleotide sequence of a cDNAdisclosed herein. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in a Cechet al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, an mRNA encoding a polypeptide of the invention can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, expression of a polypeptide of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93: 14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc.Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which may combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNAse H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996), supra).The synthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al. (1989) Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′DNA segment and a 3′ PNA segment (Peterser et al.(1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g.,Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

II. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise antibodies directed against apolypeptide of the invention. In one embodiment, the native polypeptidecan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, polypeptides of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, apolypeptide of the invention can be synthesized chemically usingstandard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide of the invention includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the protein (e.g., the aminoacid sequence shown in any of SEQ ID NO:6, 7, 9, 10, 19, 20, 21, 22 or23, which include fewer amino acids than the full length protein, andexhibit at least one activity of the corresponding full-length protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the corresponding protein. A biologicallyactive portion of a protein of the invention can be a polypeptide whichis, for example, 10, 25, 50, 100 or more amino acids in length.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of the nativeform of a polypeptide of the invention.

Preferred polypeptides have the amino acid sequence of SEQ ID NO:6, 7,8, 9, 10, 19, 20, 21, 22 or 23. Other useful proteins are substantiallyidentical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%,or 99%) to any of SEQ ID NO:6, 7, 8, 9, 10, 19, 20, 21, 22 or 23, andretain the functional activity of the protein of the correspondingnaturally-occurring protein yet differ in amino acid sequence due tonatural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (, % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment, the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the CGC sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art and include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. For a further description of FASTA parameters, seehttp://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2, the contentsof which are incorporated herein by reference.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The invention also provides chimeric or fusion proteins. As used herein,a “chimeric protein” or “fusion protein” comprises all or part(preferably biologically active) of a polypeptide of the inventionoperably linked to a heterologous polypeptide (i.e., a polypeptide otherthan the same polypeptide of the invention). Within the fusion protein,the term “operably linked” is intended to indicate that the polypeptideof the invention and the heterologous polypeptide are fused in-frame toeach other. The heterologous polypeptide can be fused to the N-terminusor C-terminus of the polypeptide of the invention.

One useful fusion protein is a GST fusion protein in which thepolypeptide of the invention is fused to the C-terminus of GSTsequences. Such fusion proteins can facilitate the purification of arecombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus. For example, the native signal sequence of apolypeptide of the invention can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Sambrook et al., supra) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which all or part of a polypeptide of the invention isfused to sequences derived from a member of the immunoglobulin proteinfamily. The immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a ligand (soluble ormembrane-bound) and a protein on the surface of a cell (receptor), tothereby suppress signal transduction in vivo. The immunoglobulin fusionprotein can be used to affect the bioavailability of a cognate ligand ofa polypeptide of the invention. Inhibition of ligand/receptorinteraction may be useful therapeutically, both for treatingproliferative and differentiative disorders and for modulating (e.g.,promoting or inhibiting) cell survival. Moreover, the immunoglobulinfusion proteins of the invention can be used as immunogens to produceantibodies directed against a polypeptide of the invention in a subject,to purify ligands and in screening assays to identify molecules whichinhibit the interaction of receptors with ligands.

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence of a polypeptide of the invention (SEQ ID NO:4 or 17)can be used to facilitate secretion and isolation of the secretedprotein or other proteins of interest. Signal sequences are typicallycharacterized by a core of hydrophobic amino acids which are generallycleaved from the mature protein during secretion in one or more cleavageevents. Such signal peptides contain processing sites that allowcleavage of the signal sequence from the mature proteins as they passthrough the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as to thesignal sequence itself and to the polypeptide in the absence of thesignal sequence (i.e., the cleavage products). In one embodiment, anucleic acid sequence encoding a signal sequence of the invention can beoperably linked in an expression vector to a protein of interest, suchas a protein which is ordinarily not secreted or is otherwise difficultto isolate. The signal sequence directs secretion of the protein, suchas from a eukaryotic host into which the expression vector istransformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by art recognized methods. Alternatively, the signal sequence canbe linked to the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

In another embodiment, the signal sequences of the present invention canbe used to identify regulatory sequences, e.g., promoters, enhancers,repressors. Since signal sequences are the most amino-terminal sequencesof a peptide, it is expected that the nucleic acids which flank thesignal sequence on its amino-terminal side will be regulatory sequenceswhich affect transcription. Thus, a nucleotide sequence which encodesall or a portion of a signal sequence can be used as a probe to identifyand isolate signal sequences and their flanking regions, and theseflanking regions can be studied to identify regulatory elements therein.

The present invention also pertains to variants of the polypeptides ofthe invention. Such variants have an altered amino acid sequence whichcan function as either agonists (mimetics) or as antagonists. Variantscan be generated by mutagenesis, e.g., discrete point mutation ortruncation. An agonist can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theprotein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g.,. truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening and subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with SI nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

The polypeptides of the invention can exhibit post-translationalmodifications, including, but not limited to glycosylations, (e.g.,N-linked or O-linked glycosylations), myristylations, palmitylations,acetylations and phosphorylations (e.g., serine/threonine or tyrosine).In one embodiment, the TANGO 268 polypeptide of the invention exhibitreduced levels of 0-linked glycosylation and/or N-linked glycosylationrelative to endogenously expressed TANGO 268 polypeptides. In anotherembodiment, the TANGO 268 polypeptides of the invention do not exhibitO-linked glycosylation or N-linked glycosylation.

An isolated polypeptide of the invention, or a fragment thereof, can beused as an immunogen to generate antibodies using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthpolypeptide or protein can be used or, alternatively, the inventionprovides antigenic peptide fragments for use as immunogens. Theantigenic peptide of a protein of the invention comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence of SEQ ID NO:3 or 16, and encompasses an epitope of the proteinsuch that an antibody raised against the peptide forms a specific immunecomplex with the protein.

Epitopes encompassed by the antigenic peptide are regions that arelocated on the surface of the protein, e.g., hydrophilic regions.Alternatively, epitopes encompassed by the antigenic peptides areregions that are located within the proteins, and/or epitopes exposed indenatured or partially denatured forms of the polypeptides of theinvention. FIGS. 2 and 7 are hydropathy plots of the proteins of theinvention. These plots or similar analyses can be used to identifyhydrophilic regions. In addition, an epitope can encompass, in additionto a polypeptide or polypeptides of the invention, a post-translationalmodification (e.g., glycosylation, such as, for example, N— and/orO-linked glycosylation of the polypeptide or polypeptides).

An immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal). Anappropriate immunogenic preparation can contain, for example,recombinantly expressed or chemically synthesized polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodiesdirected against a polypeptide of the invention. The term “antibody” asused herein refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site which specifically binds an antigen,such as a polypeptide of the invention, e.g., an epitope of apolypeptide of the invention. A molecule which specifically binds to agiven polypeptide of the invention is a molecule which binds thepolypeptide, but does not substantially bind other molecules in asample, e.g., a biological sample, which naturally contains thepolypeptide. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin orpapain. The invention provides polyclonal and monoclonal antibodies. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.Preferred polyclonal antibody compositions are ones that have beenselected for antibodies directed against a polypeptide or polypeptidesof the invention. Particularly preferred polyclonal antibodypreparations are ones that contain only antibodies directed against apolypeptide or polypeptides of the invention. Particularly preferredimmunogen compositions are those that contain no other human proteinssuch as, for example, immunogen compositions made using a non-human hostcell for recombinant expression of a polypeptide of the invention. Insuch a manner, the only human epitope or epitopes recognized by theresulting antibody compositions raised against this immunogen will bepresent as part of a polypeptide or polypeptides of the invention.

The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. Alternatively, antibodiesspecific for a protein or polypeptide of the invention can be selectedfor (e.g., partially purified) or purified by, e.g., affinitychromatography. For example, a recombinantly expressed and purified (orpartially purified) protein of the invention is produced as describedherein, and covalently or non-covalently coupled to a solid support suchas, for example, a chromatography column. The column can then be used toaffinity purify antibodies specific for the proteins of the inventionfrom a sample containing antibodies directed against a large number ofdifferent epitopes, thereby generating a substantially purified antibodycomposition, i.e., one that is substantially free of contaminatingantibodies. By a substantially purified antibody composition is meant,in this context, that the antibody sample contains at most only 30% (bydry weight) of contaminating antibodies directed against epitopes otherthan those on the desired protein or polypeptide of the invention, andpreferably at most 20%, yet more preferably at most 10%, and mostpreferably at most 5% (by dry weight) of the sample is contaminatingantibodies. A purified antibody composition means that at least 99% ofthe antibodies in the composition are directed against the desiredprotein or polypeptide of the invention.

At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing hybridomas is well known (see generally CurrentProtocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons,Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibodyof the invention are detected by screening the hybridoma culturesupernatants for antibodies that bind the polypeptide of interest, e.g.,using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting bybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarily determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced, forexample, using transgenic mice which are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA and IgE antibodies. For an overview of this technology for producinghuman antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol.13:65-93). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat.Nos. 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition,companies such as Abgenix, Inc. (Fremont, Calif.), can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

An antibody directed against a polypeptide of the invention (e.g.,monoclonal antibody) can be used to isolate the polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, such an antibody can be used to detect the protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the polypeptide. The antibodies can also beused diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Further, an antibody (or fragment thereof) can be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha.-interferon, .beta.-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating a therapeutic moiety to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

Accordingly, in one aspect, the invention provides substantiallypurified antibodies or fragment thereof, and non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:3 or 16, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 207180, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as PTA-225; afragment of at least 15 amino acid residues of the amino acid sequenceof SEQ ID NO:3 or 16; an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO:3 or 16, wherein thepercent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4; and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to the nucleicacid molecule consisting of SEQ ID NO:1, 2, 14, or 15 under conditionsof hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at65° C. In various embodiments, the substantially purified antibodies ofthe invention, or fragments thereof, can be human, non-human, chimericand/or humanized antibodies.

In another aspect, the invention provides non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:3 or 16, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 207180, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as PTA-225; afragment of at least 15 amino acid residues of the amino acid sequenceof SEQ ID NO:3 or 16; an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO:3 or 16, wherein thepercent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4; and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to the nucleicacid molecule consisting of SEQ ID NO:1, 2, 14, or 15 under conditionsof hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at65° C. Such non-human antibodies can be goat, mouse, sheep, horse,chicken, rabbit, or rat antibodies. Alternatively, the non-humanantibodies of the invention can be chimeric and/or humanized antibodies.In addition, the non-human antibodies of the invention can be polyclonalantibodies or monoclonal antibodies.

In still a further aspect, the invention provides monoclonal antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:3 or 16, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 207180, or the/amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as PTA-225; afragment of at least 15 amino acid residues of the amino acid sequenceof SEQ ID NO:3 or 16; an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO:3 or 16, wherein thepercent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4; and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to the nucleicacid molecule consisting of SEQ ID NO:1, 2, 14, or 15 under conditionsof hybridization of 6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at65° C. The monoclonal antibodies can be human, humanized, chimericand/or non-human antibodies.

In a particularly preferred embodiment, the substantially purifiedantibodies or fragments thereof, the non-human antibodies or fragmentsthereof, and/or the monoclonal antibodies or fragments thereof, of theinvention specifically bind to an extracellular domain of the amino acidsequence of SEQ ID NO:3 or 16. Preferably, the extracellular domain towhich the antibody, or fragment thereof, binds comprises amino acidresidues 21 to 269 of SEQ ID NO:3 or amino acid residues 22 to 267 ofSEQ ID NO:16. In an alternative embodiment, the extracellular domain towhich the substantially purified antibody binds comprises animmunoglobulin-like domain. In one aspect, such an immunoglobulin-likedomain comprises amino acid residues 48 to 88 or 134 to 180 of SEQ IDNO:3 or amino acid residues 49 to 89 or 135 to 181 of SEQ ID NO:16.

Any of the antibodies of the invention can be conjugated to atherapeutic moiety or to a detectable substance. Non-limiting examplesof detectable substances that can be conjugated to the antibodies of theinvention are an enzyme, a prosthetic group, a fluorescent material, aluminescent material, a bioluminescent material, and a radioactivematerial.

The invention also provides a kit containing an antibody of theinvention conjugated to a detectable substance, and instructions foruse. Still another aspect of the invention is a pharmaceuticalcomposition comprising an antibody of the invention and apharmaceutically acceptable carrier. In preferred embodiments, thepharmaceutical composition contains an antibody of the invention, atherapeutic moiety, and a pharmaceutically acceptable carrier.

Still another aspect of the invention is a method of making an antibodythat specifically recognizes GPVI, the method comprising immunizing amammal with a polypeptide. The polypeptide used as an immungen comprisesan amino acid sequence selected from the group consisting of: the aminoacid sequence of SEQ ID NO:3 or 16, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as AccessionNumber 207180, or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with ATCC as PTA-225; a fragment of at least 15amino acid residues of the amino acid sequence of SEQ ID NO:3 or 16; anamino acid sequence which is at least 95% identical to the amino acidsequence of SEQ ID NO:3 or 16, wherein the percent identity isdetermined using the ALIGN program of the GCG software package with aPAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4; and an amino acid sequence which is encoded by a nucleicacid molecule which hybridizes to the nucleic acid molecule consistingof SEQ ID NO:1, 2, 14, or 15 under conditions of hybridization of 6×SSCat 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. After immunization,a sample is collected from the mammal that contains an antibody thatspecifically recognizes GPVI. Preferably, the polypeptide isrecombinantly produced using a non-human host cell. Optionally, theantibodies can be further purified from the sample using techniques wellknown to those of skill in the art. The method can further compriseproducing a monoclonal antibody-producing cell from the cells of themammal. Optionally, antibodies are collected from the antibody-producingcell.

In instances wherein the antibody is to be utilized as a therapeutic,characterization of the antibody can routinely be assayed andascertained via the methods presented herein. For example, the fact thatplatelets are readily available, coupled with the availability ofmultiple assays for platelet function provide for routine testing andanalysis (e.g., for in vitro testing and analysis) of such antibodies.

For example, the in vivo pharmacodynamic characterization of anti-TANGO268 antibodies can be facilitated via the availability of variousplatelet assays (e.g., prolongation of bleeding time, quantitativemeasurement of TANGO 268 receptor blockade, inhibition of ex vivoplatelet aggregation) such as those described herein that can becorrelated with each other to permit more effective assessment of amodulator's functional consequences. The correlation available for suchassays, therefore, allows for the in vitro characterization of ananti-TANGO 268 antibody to more directly apply to the measurement of theantibody's therapeutic effect.

In addition to utilizing the availability of platelets and plateletassays for assessing the therapeutic efficacy, including clinicalefficacy, of an anti-TANGO 268 antibody, this availability can also beutilized for preclinical drug development aspects such as determiningantibody dosage response, toxicology, magnitude of effect (e.g.,magnitude of initial effect and magnitude of effect's duration),function, specificity (e.g., specificity with respect to particularplatelet functions), receptor specificity, and species specificity(which, in turn, can identify appropriate animal models for pharmacologystudies).

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptide ofthe invention (or a portion thereof). As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operably linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the invention is intended to include such otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic (e.g., E.coli) or eukaryotic cells (e.g., insect cells (using baculovirusexpression vectors), yeast cells or mammalian cells). Suitable hostcells are discussed further in Goeddel, supra. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET ld vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident )prophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the mouse hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the beta-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al. (Reviews—Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

In another embodiment, the expression characteristics of an endogenous(e.g., TANGO 268 genes) within a cell, cell line or microorganism may bemodified by inserting a DNA regulatory element heterologous to theendogenous gene of interest into the genome of a cell, stable cell lineor cloned microorganism such that the inserted regulatory element isoperatively linked with the endogenous gene (e.g., TANGO 268 genes) andcontrols, modulates or activates. For example, endogenous TANGO 268geneswhich are normally “transcriptionally silent”, i.e., a TANGO 268 geneswhich is normally not expressed, or are expressed only at very lowlevels in a cell line or microorganism, may be activated by inserting aregulatory element which is capable of promoting the expression of anormally expressed gene product in that cell line or microorganism.Alternatively, transcriptionally silent, endogenous TANGO 268 genes maybe activated by insertion of a promiscuous regulatory element that worksacross cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked withand activates expression of endogenous TANGO 268 genes, usingtechniques, such as targeted homologous recombination, which are wellknown to those of skill in the art, and described e.g., in Chappel, U.S.Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May 16,1991.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a polypeptide of the invention.Accordingly, the invention further provides methods for producing apolypeptide of the invention using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding apolypeptide of the invention has been introduced) in a suitable mediumsuch that the polypeptide is produced. In another embodiment, the methodfurther comprises isolating the polypeptide from the medium or the hostcell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which asequence encoding a polypeptide of the invention has been introduced.Such host cells can then be used to create non-human transgenic animalsin which exogenous sequences encoding a polypeptide of the inventionhave been introduced into their genome or homologous recombinant animalsin which endogenous encoding a polypeptide of the invention sequenceshave been altered. Such animals are useful for studying the functionand/or activity of the polypeptide and for identifying and/or evaluatingmodulators of polypeptide activity. As used herein, a “transgenicanimal” is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, an “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous gene has been altered byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingnucleic acid encoding a polypeptide of the invention (or a homologuethereof) into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection, retroviral infection, and allowing the oocyte to developin a pseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, 4,873,191 and in Hogan, Manipulatingthe Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1986). Similar methods are used for production of othertransgenic animals. A transgenic founder animal can be identified basedupon the presence of the transgene in its genome and/or expression ofmRNA encoding the transgene in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying thetransgene can further be bred to other transgenic animals carrying othertransgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptide of theinvention into which a deletion, addition or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the gene. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous gene is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous protein). In the homologous recombination vector, the alteredportion of the gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the gene to allow for homologous recombination to occurbetween the exogenous gene carried by the vector and an endogenous genein an embryonic stem cell. The additional flanking nucleic acidsequences are of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced gene has homologously recombined with the endogenous geneare selected (see, e.g., Li et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford,1987) pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Biolfechnology 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/toxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies (also referredto herein as “active compounds”) of the invention can be incorporatedinto pharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more additionalactive compounds.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mgfkg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

Preferably, administration of the TANGO 268 modulator is at or near thesite of the cells or tissue to be treated, e.g., administration is at ornear the site of a platelet aggregation-induced disorder such as one ofthose described herein.

In certain embodiments, the TANGO 268 modulator is administered orco-administered with at least one other desirable agent, e.g., heparinor aspirin.

In certain instances, it is preferred that administration of a TANGO 268modulator comprises an initial bolus followed by continuous infusion.For example, such instances will generally include those wherein themodulator exhibits appreciable reversibility in platelet binding, as,e.g., assayed via the techniques described herein.

In one example, presented by way of illustration and not by way oflimitation, a dosage and administration regimen for treatment ofischemic heart disease or thromboses comprises: in patients undergoingpercutaneous coronary angioplasty (PCA), the TANGO 268 modulator (e.g.,anti-TANGO 268 antibody) is administered as a 0.25 mg/kg IV bolus plusinfusion of 10 μg/min or 0.125 μg/kg/min (this can, alternatively, beperformed in conjunction with heparin and aspirin) for 12 hours. Inpatients with refractory unstable angina in whom PCA is planned within24 hours, the bolus and infusion are given 18-24 hours before theprocedure and the infusion is continued for 1 hour or 12 hours after theprocedure.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the ken of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology); c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express proteins (e.g., via a recombinant expression vectorin a host cell in gene therapy applications), to detect mRNA (e.g., in abiological sample) or a genetic lesion, and to modulate activity of apolypeptide of the invention. In addition, the polypeptides of theinvention can be used to screen drugs or compounds which modulateactivity or expression of a polypeptide of the invention as well as totreat disorders characterized by insufficient or excessive production ofa protein of the invention or production of a form of a protein of theinvention which has decreased or aberrant activity compared to the wildtype protein. In addition, the antibodies of the invention can be usedto detect and isolate a protein of the invention or to modulate activityof a protein of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to polypeptide of the invention or have a stimulatory orinhibitory effect on, for example, expression or activity of apolypeptide of the invention.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of a polypeptide of the invention or biologicallyactive portion thereof. The test compounds of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of a polypeptide of the invention, or abiologically active portion thereof, on the cell surface is contactedwith a test compound and the ability of the test compound to bind to thepolypeptide determined. The cell, for example, can be a yeast cell or acell of mammalian origin. Determining the ability of the test compoundto bind to the polypeptide can be accomplished, for example, by couplingthe test compound with a radioisotope or enzymatic label such thatbinding of the test compound to the polypeptide or biologically activeportion thereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish perbxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. In a preferred embodiment, the assaycomprises contacting a cell which expresses a membrane-bound form of apolypeptide of the invention, or a biologically active portion thereof,on the cell surface with a known compound which binds the polypeptide toform an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith the polypeptide, wherein determining the ability of the testcompound to interact with the polypeptide comprises determining theability of the test compound to preferentially bind to the polypeptideor a biologically active portion thereof as compared to the knowncompound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of a polypeptide ofthe invention, or a biologically active portion thereof, on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of thepolypeptide or biologically active portion thereof. Determining theability of the test compound to modulate the activity of the polypeptideor a biologically active portion thereof can be accomplished, forexample, by determining the ability of the polypeptide protein to bindto or interact with a target molecule.

Determining the ability of a polypeptide of the invention to bind to orinteract with a target molecule can be accomplished by one of themethods described above for determining direct binding. As used herein,a “target molecule” is a molecule with which a selected polypeptide(e.g., a polypeptide of the invention) binds or interacts with innature, for example, a molecule on the surface of a cell which expressesthe selected protein, a molecule on the surface of a second cell, amolecule in the extracellular milieu, a molecule associated with theinternal surface of a cell membrane or a cytoplasmic molecule. A targetmolecule can be a polypeptide of the invention or some other polypeptideor protein. For example, a target molecule can be a component of asignal transduction pathway which facilitates transduction of anextracellular signal (e.g., a signal generated by binding of a compoundto a polypeptide of the invention) through the cell membrane and intothe cell or a second intercellular protein which has catalytic activityor a protein which facilitates the association of downstream signalingmolecules with a polypeptide of the invention. Determining the abilityof a polypeptide of the invention to bind to or interact with a targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (e.g., intracellular Ca²⁺, diacylglycerol, IP3, etc.), detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (e.g., a regulatory elementthat is responsive to a polypeptide of the invention operably linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a cellular response, for example, cellular differentiation, orcell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a polypeptide of the invention orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to bind to the polypeptide orbiologically active portion thereof. Binding of the test compound to thepolypeptide can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting thepolypeptide of the invention or biologically active portion thereof witha known compound which binds the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the test compound topreferentially bind to the polypeptide or biologically active portionthereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting a polypeptide of the invention or biologically active portionthereof with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of thepolypeptide or biologically active portion thereof. Determining theability of the test compound to modulate the activity of the polypeptidecan be accomplished, for example, by determining the ability of thepolypeptide to bind to a target molecule by one of the methods describedabove for determining direct binding. In an alternative embodiment,determining the ability of the test compound to modulate the activity ofthe polypeptide can be accomplished by determining the ability of thepolypeptide of the invention to further modulate the target molecule.For example, the catalytic/enzymatic activity of the target molecule onan appropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting apolypeptide of the invention or biologically active portion thereof witha known compound which binds the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the polypeptide topreferentially bind to or modulate the activity of a target molecule.

The cell-free assays of the present invention are amenable to use ofboth a soluble form or the membrane-bound form of a polypeptide of theinvention. In the case of cell-free assays comprising the membrane-boundform of the polypeptide, it may be desirable to utilize a solubilizingagent such that the membrane-bound form of the polypeptide is maintainedin solution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-octylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either the polypeptide ofthe invention or its target molecule to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound tothe polypeptide, or interaction of the polypeptide with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase fusionproteins or glutathione-S-transferase fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or A polypeptide of the invention, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtitre plate wells are washed to remove any unboundcomponents and complex formation is measured either directly orindirectly, for example, as described above. Alternatively, thecomplexes can be dissociated from the matrix, and the level of bindingor activity of the polypeptide of the invention can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either thepolypeptide of the invention or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylatedpolypeptide of the invention or target molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well known in theart (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with the polypeptide ofthe invention or target molecules but which do not interfere withbinding of the polypeptide of the invention to its target molecule canbe derivatized to the wells of the plate, and unbound target orpolypeptide of the invention trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with thepolypeptide of the invention or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the polypeptide of the invention or target molecule.

In another embodiment, modulators of expression of a polypeptide of theinvention are identified in a method in which a cell is contacted with acandidate compound and the expression of the selected mRNA or protein(i.e., the mRNA or protein corresponding to a polypeptide or nucleicacid of the invention) in the cell is determined. The level ofexpression of the selected mRNA or protein in the presence of thecandidate compound is compared to the level of expression of theselected mRNA or protein in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of expressionof the polypeptide of the invention based on this comparison. Forexample, when expression of the selected mRNA or protein is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of the selected mRNA or protein expression. Alternatively,when expression of the selected mRNA or protein is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor of theselected mRNA or protein expression. The level of the selected mRNA orprotein expression in the cells can be determined by methods describedherein.

In yet another aspect of the invention, a polypeptide of the inventionscan be used as “bait proteins” in a two-hybrid assay or three hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with the polypeptide of theinvention and modulate activity of the polypeptide of the invention.Such binding proteins are also likely to be involved in the propagationof signals by the polypeptide of the inventions as, for example,upstream or downstream elements of a signaling pathway involving thepolypeptide of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, nucleic acid molecules described herein orfragments thereof, can be used to map the location of the correspondinggenes on a chromosome. The mapping of the sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, genes can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp in length) from the sequence of a gene of theinvention. Computer analysis of the sequence of a gene of the inventioncan be used to rapidly select primers that do not span more than oneexon in the genomic DNA, thus complicating the amplification process.These primers can then be used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the gene sequences will yield anamplified fragment. For a review of this technique, see D'Eustachio etal. ((1983) Science 220:919-924).

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the nucleicacid sequences of the invention to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa gene to its chromosome include in situ hybridization (described in Fanet al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening withlabeled flow-sorted chromosomes (CITE), and pre-selection byhybridization to chromosome specific cDNA libraries. Fluorescence insitu hybridization (FISH) of a DNA sequence to a metaphase chromosomalspread can further be used to provide a precise chromosomal location inone step. For a review of this technique, see Verma et al., (HumanChromosomes: A Manual of Basic Techniques (Pergamon Press, New York,1988)).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with a gene of the inventioncan be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

In the instant case, the human gene for GPVI was mapped on radiationhybrid panels to the long arm of chromosome 19, in the region 19q13.This region is syntenic to mouse chromosome 7. Multiple members of theimmunoglobulin superfamily, including killer cell inhibitory receptors,immunoglobulin-like transcripts (ILTI, 2, 3), the gp49b family and theFc_receptor (CD89) also map to this region of the human chromosome.These various receptors differ considerably with respect to function andexpression and it may be hypothesized that functional differentiationoccurred after duplication of a common ancestral gene.

The mouse gene for GPVI was mapped using the T31 Mouse/Hamster RadiationHybrid (McCarthy, Terrett et al. 1997). PCR Amplification used thefollowing mouse primers: forward primer 5′-CTGTAGCTGTTTTCAGACACACC-3′(SEQ ID NO:31) and reverse primer 5′-CCATCACCTCTTTCTGGTTAC-3′ (SEQ IDNO:32). All PCRs were performed with an annealing temperature of 52_Cand extension times of 50 s (72_C) and for 35 cycles, with a finalextension of 5 minutes on an MJ Research Peltier PCT-225 Thermal Cycler.

2. Tissue Typing

The nucleic acid sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the nucleic acid sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The nucleic acid sequences of the invention uniquely represent portionsof the human genome. Allelic variation occurs to some degree in thecoding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency at about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1 or 14can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:1 or 14 are used, a more appropriate number of primers forpositive individual identification would be 500-2,000.

If a panel of reagents from the nucleic acid sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial Gene Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the nucleic acid sequencesof the invention or portions thereof, e.g., fragments derived fromnoncoding regions having a length of at least 20 or 30 bases.

The nucleic acid sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such probes can be used to identify tissue byspecies and/or by organ type.

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningexpression of a polypeptide or nucleic acid of the invention and/oractivity of a polypeptide of the invention, in the context of abiological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrantexpression or activity of a polypeptide of the invention. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith aberrant expression or activity of a polypeptide of the invention.For example, mutations in a gene of the invention can be assayed in abiological sample. Such assays can be used for prognostic or predictivepurpose to thereby prophylactically treat an individual prior to theonset of a disorder characterized by or associated with aberrantexpression or activity of a polypeptide of the invention.

Another aspect of the invention provides methods for expression of anucleic acid or polypeptide of the invention or activity of apolypeptide of the invention in an individual to thereby selectappropriate therapeutic or prophylactic agents for that individual(referred to herein as “pharmacogenomics”). Pharmacogenomics allows forthe selection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the genotype of the individual(e.g., the genotype of the individual examined to determine the abilityof the individual to respond to a particular agent).

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof a polypeptide of the invention in clinical trials. These and otheragents are described in further detail in the following sections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid of the invention in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of theinvention such that the presence of a polypeptide or nucleic acid of theinvention is detected in the biological sample. A preferred agent fordetecting mRNA or genomic DNA encoding a polypeptide of the invention isa labeled nucleic acid probe capable of hybridizing to mRNA or genomicDNA encoding a polypeptide of the invention. The nucleic acid probe canbe, for example, a full-length cDNA, such as the nucleic acid of SEQ IDNO:1, 2, 14 or 15, or a portion thereof, such as an oligonucleotide ofat least 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to amRNA or genomic DNA encoding a polypeptide of the invention. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

A preferred agent for detecting a polypeptide of the invention is anantibody capable of binding to a polypeptide of the invention,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect mRNA, protein, or genomic DNA in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof mRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of a polypeptide of the invention includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of genomic DNA include Southern hybridizations. Furthermore,in vivo techniques for detection of a polypeptide of the inventioninclude introducing into a subject a labeled antibody directed againstthe polypeptide. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting a polypeptide of theinvention or mRNA or genomic DNA encoding a polypeptide of theinvention, such that the presence of the polypeptide or mRNA or genomicDNA encoding the polypeptide is detected in the biological sample, andcomparing the presence of the polypeptide or mRNA or genomic DNAencoding the polypeptide in the control sample with the presence of thepolypeptide or mRNA or genomic DNA encoding the polypeptide in the testsample.

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid of the invention in a biological sample (atest sample). Such kits can be used to determine if a subject issuffering from or is at increased risk of developing a disorderassociated with aberrant expression of a polypeptide of the invention asdiscussed, for example, n sections above relating to uses of thesequences of the invention.

For example, kits can be used to determine if a subject is sufferingfrom or is at increased risk of disorders such as immunologicaldisorders, (e.g. thrombocytopenia and platelet disorders), liverdisorders, cerebral vascular diseases (e.g., stroke and ischemia),venous thromboembolisme diseases (e.g., diseases involving leg swelling,pain and ulceration, pulmonary embolism, abdominal venous thrombosis),coronary diseases (e.g., cardiovascular diseases including unstableangina, acute myocardial infarction, coronary artery disease, coronaryrevascularization, ventricular thromboembolism, atherosclerosis,coronary artery disease, and plaque formation), metastatic cancers(e.g., the metastasis of cancerous colon and liver cells) and embryonicdisorders, which are associated with aberrant TANGO 268 expression. Thekit, for example, can comprise a labeled compound or agent capable ofdetecting the polypeptide or mRNA encoding the polypeptide in abiological sample and means for determining the amount of thepolypeptide or mRNA in the sample (e.g., an antibody which binds thepolypeptide or an oligonucleotide probe which binds to DNA or mRNAencoding the polypeptide). Kits can also include instructions forobserving that the tested subject is suffering from or is at risk ofdeveloping a disorder associated with aberrant expression of thepolypeptide if the amount of the polypeptide or mRNA encoding thepolypeptide is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide of the invention; and, optionally, (2) a second, differentantibody which binds to either the polypeptide or the first antibody andis conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptide of theinvention or (2) a pair of primers useful for amplifying a nucleic acidmolecule encoding a polypeptide of the invention. The kit can alsocomprise, e.g., a buffering agent, a preservative, or a proteinstabilizing agent. The kit can also comprise components necessary fordetecting the detectable agent (e.g., an enzyme or a substrate). The kitcan also contain a control sample or a series of control samples whichcan be assayed and compared to the test sample contained. Each componentof the kit is usually enclosed within an individual container and all ofthe various containers are within a single package along withinstructions for observing whether the tested subject is suffering fromor is at risk of developing a disorder associated with aberrantexpression of the polypeptide.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of a polypeptide of the invention. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with aberrant expression oractivity of a polypeptide of the invention, e.g., an immunologicdisorder, or embryonic disorders. Alternatively, the prognostic assayscan be utilized to identify a subject having or at risk for developingsuch a disease or disorder. Thus, the present invention provides amethod in which a test sample is obtained from a subject and apolypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the inventionis detected, wherein the presence of the polypeptide or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant expression or activity of thepolypeptide. As used herein, a “test sample” refers to a biologicalsample obtained from a subject of interest. For example, a test samplecan be a biological fluid (e.g., serum), cell sample, or tissue.

The prognostic assays described herein, for example, can be used toidentify a subject having or at risk of developing disorders such asdisorders discussed, for example, in sections above relating to uses ofthe sequences of the invention. For example, such disorders can includeimmunological disorders, (e.g. thrombocytopenia and platelet disorders),liver disorders, cerebral vascular diseases (e.g., stroke and ischemia),venous thromboembolisme diseases (e.g., diseases involving leg swelling,pain and ulceration, pulmonary embolism, abdominal venous thrombosis),coronary diseases (e.g., cardiovascular diseases including unstableangina, acute myocardial infarction, coronary artery disease, coronaryrevascularization, ventricular thromboembolism, atherosclerosis,coronary artery disease, and plaque formation), metastatic cancers(e.g., the metastasis of cancerous colon and liver cells) andprogression to such metastatic tumors, developmental disorders andembryonic disorders, which are associated with aberrant TANGO 268expression.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant expression or activity of a polypeptide of theinvention. For example, such methods can be used to determine whether asubject can be effectively treated with a specific agent or class ofagents (e.g., agents of a type which decrease activity of thepolypeptide). Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant expression or activity of apolypeptide of the invention in which a test sample is obtained and thepolypeptide or nucleic acid encoding the polypeptide is detected (e.g.,wherein the presence of the polypeptide or nucleic acid is diagnosticfor a subject that can be administered the agent to treat a disorderassociated with aberrant expression or activity of the polypeptide).

The methods of the invention can also be used to detect genetic lesionsor mutations in a gene of the invention, thereby determining if asubject with the lesioned gene is at risk for a disorder characterizedaberrant expression or activity of a polypeptide of the invention. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesion ormutation characterized by at least one of an alteration affecting theintegrity of a gene encoding the polypeptide of the invention, or themis-expression of the gene encoding the polypeptide of the invention.For example, such genetic lesions or mutations can be detected byascertaining the existence of at least one of: 1) a deletion of one ormore nucleotides from the gene; 2) an addition of one or morenucleotides to the gene; 3) a substitution of one or more nucleotides ofthe gene; 4) a chromosomal rearrangement of the gene; 5) an alterationin the level of a messenger RNA transcript of the gene; 6) an aberrantmodification of the gene, such as of the methylation pattern of thegenomic DNA; 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of the gene; 8) a non-wild type level of a theprotein encoded by the gene; 9) an allelic loss of the gene; and 10) aninappropriate post-translational modification of the protein encoded bythe gene. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in agene.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a gene (see, e.g.,Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to the selected gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a selected gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No.5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations can be identified by hybridizinga sample and control nucleic acids, e.g., DNA or RNA, to high densityarrays containing hundreds or thousands of oligonucleotides probes(Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996)Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin et al., supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the selected gene anddetect mutations by comparing the sequence of the sample nucleic acidswith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin etal. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in a selected gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the technique of “mismatch cleavage” entailsproviding heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type sequence with potentially mutant RNA or DNAobtained from a tissue sample. The double-stranded duplexes are treatedwith an agent which cleaves single-stranded regions of the duplex suchas which will exist due to basepair mismatches between the control andsample strands. RNA/DNA duplexes can be treated with RNase to digestmismatched regions, and DNA/DNA hybrids can be treated with S 1 nucleaseto digest mismatched regions.

In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g., Cottonet al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in cDNAs obtained from samples ofcells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a selectedsequence, e.g., a wild-type sequence, is hybridized to a cDNA or otherDNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, e.g., U.S.Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). Single-stranded DNA fragments of sample and control nucleicacids will be denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, and theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a 'GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6: 1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a gene encoding apolypeptide of the invention. Furthermore, any cell type or tissue,e.g., chondrocytes, in which the polypeptide of the invention isexpressed may be utilized in the prognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onactivity or expression of a polypeptide of the invention as identifiedby a screening assay described herein can be administered to individualsto treat (prophylactically or therapeutically) disorders associated withaberrant activity of the polypeptide. In conjunction with suchtreatment, the pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) of the individual may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of a polypeptide of the invention,expression of a nucleic acid of the invention, or mutation content of agene of the invention in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of a polypeptide of the invention, expression of anucleic acid encoding the polypeptide, or mutation content of a geneencoding the polypeptide in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used toapply genotyping of polymorphic alleles encoding drug-metabolizingenzymes to the identification of an individual's drug responsivenessphenotype. This knowledge, when applied to dosing or drug selection, canavoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with amodulator of activity or expression of the polypeptide, such as amodulator identified by one of the exemplary screening assays describedherein.

4. Monitoring of TANGO 268 Modulator Effects

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of a polypeptide of the invention (e.g., theability to modulate aberrant cell proliferation chemotaxis, and/ordifferentiation) can be applied in basic drug screening, preclinicalstudies, clinical trials and during therapeutic treatment regimens.

For example, the effectiveness of an agent, as determined by a screeningassay as described herein, to increase gene expression, protein levelsor protein activity, can be monitored in clinical trials of subjectsexhibiting decreased gene expression, protein levels, or proteinactivity. Alternatively, the effectiveness of an agent, as determined bya screening assay, to decrease gene expression, protein levels orprotein activity, can be monitored in clinical trials of subjectsexhibiting increased gene expression, protein levels, or proteinactivity. In such clinical trials, expression or activity of apolypeptide of the invention and preferably, that of other polypeptidethat have been implicated in for example, a cellular proliferationdisorder, can be used as a marker of the immune responsiveness of aparticular cell.

For example, and not by way of limitation, genes, including those of theinvention, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates activity or expressionof a polypeptide of the invention (e.g., as identified in a screeningassay described herein) can be identified. Thus, to study the effect ofagents on cellular proliferation disorders, for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of a gene of the invention and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of a gene of the invention or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of the polypeptide or nucleic acidof the invention in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thelevel the of the polypeptide or nucleic acid of the invention in thepost-administration samples; (v) comparing the level of the polypeptideor nucleic acid of the invention in the pre-administration sample withthe level of the polypeptide or nucleic acid of the invention in thepost-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of the polypeptide to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of the polypeptide to lower levels thandetected, i.e., to decrease the effectiveness of the agent.

TANGO 268 is expressed on the surface of platelets. As such, a cellularand therapeutic target of modulators of TANGO 268 (e.g., an anti-TANGO268 antibody) is readily available for testing and analysis (e.g., forin vitro testing and analysis). This coupled with the availability ofseveral different relevant platelet assays (see above) provides anunusual drug development opportunity for TANGO 268 modulators. Forexample, the in vivo pharmacodynamic characterization of TANGO 268modulators can be facilitated via the availability of various plateletassays (e.g., prolongation of bleeding time, quantitative measurement ofTANGO 268 receptor blockade, inhibition of ex vivo platelet aggregation)that can be correlated with each other to permit more effectiveassessment of a modulator's functional consequences. The correlationavailable for such assays, therefore, allows for the in vitrocharacterization of a TANGO 268 modulator to more directly apply to themeasurement of the modulator's therapeutic effect.

In addition to utilizing the availability of platelets and plateletassays for assessing the therapeutic efficacy, including clinicalefficacy, of a TANGO 268 modulator, this availability can also beutilized for preclinical drug development aspects such as determiningmodulator dosage response, toxicology, magnitude of effect (e.g.,magnitude of initial effect and magnitude of effect's duration),function, specificity (e.g., specificity with respect to particularplatelet functions), receptor specificity, and species specificity(which, in turn, can identify appropriate animal models for pharmacologystudies).

In one embodiment, therefore, a method of the invention includes amethod for identifying a TANGO 268 modulator, (e.g., an anti-TANGO 268antibody), comprising: incubating a platelet (preferably humanplatelet)-rich sample with a compound and a platelet agonist (e.g., ADP,epinephrine, thrombin, collagen), and assaying platelet aggregation,such that if platelet aggregation in the sample differs from that of acorresponding platelet-rich sample incubated with the platelet agonistin the absence of the compound, then a modulator of TANGO 268 plateletaggregation is identified. In a variation of this embodiment, the sampleis incubated with the compound prior to addition and concurrentincubation with the platelet agonist.

In another embodiment, a method of the invention includes a method formonitoring the clinical efficacy of a TANGO 268 modulator (or theeffectiveness of treatment with a TANGO 268 modulator), comprising:incubating a patient sample comprising platelets (a platelet-richsample, e.g., one containing approximately 200,000-/300,000 plateletsper ml³) with a platelet agonist, measuring the level of plateletaggregation in the sample, and comparing the level obtained with that ofa corresponding control platelet sample, wherein the patient sample isobtained from a patient to whom a TANGO 268 modulator has beenadministered, and the control platelet sample is one that has beenincubated with the platelet agonist but has not been treated with theTANGO 268 modulator. In instances wherein the aggregation level obtainedin the patient sample is lower than that of the control sample, themonitoring of the clinical efficacy of the TANGO 268 modulator (or theeffectiveness of treatment with the TANGO 268 modulator) is confirmed.

In yet another embodiment, a method of the invention includes a methodfor determining the therapeutic dosage of a TANGO 268 modulator to beadministered to an individual in need of treatment for a TANGO268-related disorder, comprising: administering a dose of a TANGO 268modulator to a non-human animal model of a TANGO 268-related disorder,and assaying TANGO 268 function and/or assaying a symptom of the TANGO268-related disorder in the animal, so that if TANGO 268 function and/orsymptom in the animal is modulated in a manner that more closelyresembles a corresponding animal not exhibiting the TANGO 268 disorder,a therapeutic dosage of the TANGO 268 modulator is determined, e.g., byextrapolating to the corresponding dosage in a human.

In a particular embodiment of such a method, platelet function and/oraggregation is assayed via, e.g., techniques such as those presentedherein. Further, the animal model can be, e.g., one of the animal modelsdescribed herein.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant expression or activity ofa polypeptide of the invention, as discussed, for example, in sectionsabove relating to uses of the sequences of the invention. For example,disorders characterized by aberrant expression or activity of thepolypeptides of the invention include immunologic disorders,developmental disorders, embryonic disorders, liver disorders, cerebralvascular diseases (e.g., stroke and ischemia), venous thromboembolismediseases (e.g., diseases involving leg swelling, pain and ulceration,pulmonary embolism, abdominal venous thrombosis), coronary diseases(e.g., cardiovascular diseases including unstable angina, acutemyocardial infarction, coronary artery disease, coronaryrevascularization, ventricular thromboembolism, atherosclerosis,coronary artery disease, and plaque formation), and metastatic cancers(e.g., the metastasis of cancerous colon and liver cells). The nucleicacids, polypeptides, and modulators thereof of the invention can be usedto treat immunologic diseases and disorders (e.g., platelet disorders),embryonic disorders liver disorders, cerebral vascular diseases (e.g.,stroke and ischemia), venous thromboembolisme diseases (e.g., diseasesinvolving leg swelling, pain and ulceration, pulmonary embolism,abdominal venous thrombosis), thrombotic disorders (e.g., thromboticocclusion of coronary arteries), coronary diseases (e.g., cardiovasculardiseases, including unstable angina pectoris, myocardial infarction,acute myocardial infarction, coronary artery disease, coronaryrevascularization, coronary restenosis, ventricular thromboembolism,atherosclerosis, coronary artery disease (e.g., arterial occulsivedisorders), and plaque formation, cardiac ischemia, includingcomplications related to coronary procedures, such as percutaneouscoronary artery angioplasty (balloon angioplasty) procedures). Withrespect to coronary procedures, such modulation can be achieved viaadministration of GPVI modulators prior to, during, or subsequent to theprocedure. In a preferred embodiment, such administration can beutilized to prevent acture cardiac ischemia following angioplasty. andmetastatic cancers (e.g., the metastasis of cancerous colon and livercells), as well as other disorders described herein.

TANGO 268 nucleic acids, proteins and modulators thereof can, therefore,be used to modulate disorders resulting from any blood vessel insultthat can result in platelet aggregation. Such blood vessel insultsinclude, but are not limited to, vessel wall injury, such as vesselinjuries that result in a highly thrombogenic surface exposed within anotherwise intact blood vessel e.g., vessel wall injuries that result inrelease of ADP, thrombin and/or epinephrine, fluid shear stress thatoccurs at the site of vessel narrowing, ruptures and/or tears at thesites of atherosclerotic plaques, and injury resulting from balloonangioplasty or atherectomy.

Preferably, the TANGO 268 nucleic acids, proteins and modulators (e.g.,anti-TANGO 268 antibodies) thereof do not effect initial plateletadhesion to vessel surfaces, or effect such adhesion to a relativelylesser extent than the effect on platelet-platelet aggregation, e.g.,unregulated platelet-platelet aggregation, following the initialplatelet adhesion. Further, in certain embodiments, it is preferred thatthe TANGO 268 nucleic acids, proteins and modulators (e.g., anti-TANGO268 antibodies) thereof do not effect other platelet attributes orfunctions, such as agonist-induced platelet shape change (e.g.,GPlb-vWF-mediated platelet agglutination induced by ristocetin), releaseof internal platelet granule components, activation of signaltransduction pathways or induction of calcium mobilization upon plateletactivation.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant expressionor activity of a polypeptide of the invention, by administering to thesubject an agent which modulates expression or at least one activity ofthe polypeptide. Subjects at risk for a disease which is caused orcontributed to by aberrant expression or activity of a polypeptide ofthe invention can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of aberrancy, for example, an agonist or antagonist agent canbe used for treating the subject. For example, an antagonist of a TANGO240 protein may be used to treat an arthropathic disorder, e.g.,rheumatoid arthritis. The appropriate agent can be determined based onscreening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingexpression or activity of a polypeptide of the invention for therapeuticpurposes. The modulatory method of the invention involves contacting acell with an agent that modulates one or more of the activities of thepolypeptide. An agent that modulates activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of the polypeptide, a peptide, apeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more of the biological activities of the polypeptide.Examples of such stimulatory agents include the active polypeptide ofthe invention and a nucleic acid molecule encoding the polypeptide ofthe invention that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more of the biological activitiesof the polypeptide of the invention. Examples of such inhibitory agentsinclude antisense nucleic acid molecules and antibodies. Thesemodulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g., by administeringthe agent to a subject). As such, the present invention provides methodsof treating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a polypeptide of theinvention. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) expression or activity. In another embodiment, the methodinvolves administering a polypeptide of the invention or a nucleic acidmolecule of the invention as therapy to compensate for reduced oraberrant expression or activity of the polypeptide.

Stimulation of activity is desirable in situations in which activity orexpression is abnormally low or downregulated and/or in which increasedactivity is likely to have a beneficial effect. Conversely, inhibitionof activity is desirable in situations in which activity or expressionis abnormally high or upregulated and/or in which decreased activity islikely to have a beneficial effect.

The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference.

Deposit of Clones

A clone containing a cDNA molecule encoding human TANGO 268 (cloneEpthEal Idl) was deposited with the American Type Culture Collection,10801 University Boulevard, Manassas, Va., 20110-2209, on Mar. 30, 1999as Accession Number 207180.

A clone containing a cDNA molecule encoding mouse TANGO 268 (cloneEpTm268) was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va., 20110-2209, on Jun. 14, 1999 asPTA-225.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence which isat least 45% identical to the nucleotide sequence of SEQ ID NO:1, 2, 14or 15, the cDNA insert of the plasmid deposited with the ATCC asAccession Number 207180 or PTA-225, or a complement thereof; b) anucleic acid molecule comprising a fragment of at least 300 nucleotidesof the nucleotide sequence of SEQ ID NO:1, 2, 14 or 15, the cDNA insertof the plasmid deposited with the ATCC as Accession Number 207180 orPTA-225, or a complement thereof; c) a nucleic acid molecule whichencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3or 16, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number 207180 or PTA-225;and d) a nucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or 16, or the aminoacid sequence encoded by the cDNA insert of the plasmid deposited withthe ATCC as Accession Number 207180 or PTA-225, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:3 or 16, orthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as Accession Number 207180 or PTA-225;
 2. Theisolated nucleic acid molecule of claim 1, which is selected from thegroup consisting of: a) a nucleic acid comprising the nucleotidesequence of SEQ ID NO:1, 2, 14 or 15, the cDNA insert of the plasmiddeposited with the ATCC as Accession Number 207180 or PTA-225, or acomplement thereof; and b) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:3 or 16, orthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as Accession Number 207180 or PTA-225;
 3. Thenucleic acid molecule of claim 1 further comprising vector nucleic acidsequences.
 4. The nucleic acid molecule of claim 1 further comprisingnucleic acid sequences encoding a heterologous polypeptide.
 5. A hostcell which contains the nucleic acid molecule of claim
 1. 6. The hostcell of claim 5 which is a mammalian host cell.
 7. A non-human mammalianhost cell containing the nucleic acid molecule of claim
 1. 8. Anisolated polypeptide selected from the group consisting of: a) afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:3 or 16, wherein the fragment comprises at least 15 contiguous aminoacids of SEQ ID NO:3 or 16; b) a naturally occurring allelic variant ofa polypeptide comprising the amino acid sequence of SEQ ID NO:3 or 16,or the amino acid sequence encoded by the cDNA insert of plasmidsdeposited with the ATCC as Accession Number 207180 or PTA-225, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:2 or 15, or a complementthereof under stringent conditions; and c) a polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequencewhich is at least 45% identical to a nucleic acid comprising thenucleotide sequence of SEQ ID NO:2 or 15, or at least 98% to a nucleicacid comprising the nucleotide sequence of SEQ ID NO:2 or 15, or acomplement thereof.
 9. The isolated polypeptide of claim 8 comprisingthe amino acid sequence of SEQ ID NO:3 or
 16. 10. The polypeptide ofclaim 8, further comprising heterologous amino acid sequences.
 11. Anantibody which selectively binds to a polypeptide of claim
 8. 12. Amethod for producing a polypeptide selected from the group consistingof: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:3or 16, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number 207180 or PTA-225,b) a polypeptide comprising a fragment of the amino acid sequence of SEQID NO:3 or 16, or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number 207180 orPTA-225, wherein the fragment comprises at least 15 contiguous aminoacids of SEQ ID NO:3 or 16, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession Number207180 or PTA-225; and c) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:3 or 16, orthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as Accession Number 207180 or PTA-225, whereinthe polypeptide is encoded by a nucleic acid molecule which hybridizesto a nucleic acid molecule comprising SEQ ID NO:1 or 14, or a complementthereof under stringent conditions; comprising culturing the host cellof claim 5 under conditions in which the nucleic acid molecule isexpressed.
 13. A method for detecting the presence of a polypeptide ofclaim 8 in a sample, comprising: a) contacting the sample with acompound which selectively binds to a polypeptide of claim 8; and b)determining whether the compound binds to the polypeptide in the sample.14. A method for detecting the presence of a nucleic acid molecule ofclaim 1 in a sample, comprising the steps of: a) contacting the samplewith a nucleic acid probe or primer which selectively hybridizes to thenucleic acid molecule; and b) determining whether the nucleic acid probeor primer binds to a nucleic acid molecule in the sample.
 15. A kitcomprising a compound which selectively hybridizes to a nucleic acidmolecule of claim 1 and instructions for use.
 16. A method foridentifying a compound which binds to a polypeptide of claim 8comprising the steps of: a) contacting a polypeptide, or a cellexpressing a polypeptide of claim 8 with a test compound; and b)determining whether the polypeptide binds to the test compound.
 17. Themethod of claim 16, wherein the binding of the test compound to thepolypeptide is detected by a method selected from the group consistingof: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay forTANGO 268-mediated signal transduction.
 18. A method for modulating theactivity of a polypeptide of claim 8 comprising contacting a polypeptideor a cell expressing a polypeptide of claim 8 with a compound whichbinds to the polypeptide in a sufficient concentration to modulate theactivity of the polypeptide.
 19. A method for identifying a compoundwhich modulates the activity of a polypeptide of claim 8, comprising: a)contacting a polypeptide of claim 8 with a test compound; and b)determining the effect of the test compound on the activity of thepolypeptide to thereby identify a compound which modulates the activityof the polypeptide.
 20. A method for modulating an activity of aTANG0268 polypeptide in a subject comprising administering ananti-TANG0268 antibody to the subject to thereby modulate an activity ofa TANG0268 polypeptide.
 21. The method of claim 20, wherein the TANG0268activity is selected from the group consisting of: a) the ability tomodulate the host immune response; b) the ability to modulate theproliferation, differentiation and/or activity of megakaryocytes and/orplatelets; c) the ability to modulate immunoregulatory functions; d) theability to modulate platelet morphology, migration, aggregation,degranulation and/or function; e) the ability to modulate collagenbinding to platelets; f) the ability modulate collagen-induced plateletadhesion and aggregation; g) the ability to interact with convulxin; h)the ability to bind to an antibody from a patient with idiopathicthrombocytopenic purpura (ITP); and i) the ability to associate and/orco-express with FcRγ.
 22. A method for treating a subject having adisorder associated with aberrant expression or activity of a TANGO268polypeptide, comprising administering an anti-TANGO268 antibody to thesubject, thereby treating said subject having a disorder associated withaberrant expression or activity of a TANGO0268 polypeptide.
 23. Themethod of claim 22, wherein the disorder associated with aberrantexpression or activity of a TANGO268 polypeptide is selected from thegroup consisting of: a) immunologic disorders; b) developmentaldisorders; c) embryonic disorders; d) liver disorders; e) cerebralvascular diseases; f) venous thromboembolism diseases; g) thromboticdisorders; h) coronary diseases; and i) metastatic cancers.